Bicyclic compounds

ABSTRACT

Disclosed are compounds of Formula (I) 
                         
and/or a salt thereof; wherein R is —OH or —OP(O)(OH) 2 . Also disclosed are methods of using such compounds as selective agonists for G protein-coupled receptor S1P 1 , and pharmaceutical compositions comprising such compounds. These compounds are useful in treating, preventing, or slowing the progression of diseases or disorders in a variety of therapeutic areas, such as autoimmune diseases and vascular disease.

This application is a continuation of U.S. application Ser. No.14/185,164, filed Feb. 20, 2014, which claims priority to U.S.Provisional Application No. 61/767,531 filed on Feb. 21, 2013, whosecontents are hereby incorporated by reference.

BACKGROUND Description

The present invention generally relates to bicyclic compounds useful asS1P₁ agonists. Provided herein are bicyclic compounds, compositionscomprising such compounds, and methods of their use. The inventionfurther pertains to pharmaceutical compositions comprising at least onecompound according to the invention that are useful for the treatment ofconditions related to S1P₁ modulation, such as autoimmune diseases andvascular disease.

Sphingosine-1-phosphate (SIP) is a zwitterionic lysophospholipidmetabolite of sphingosine (Sph), which in turn is derived from enzymaticcleavage of ceramides. Enzymatic phosphorylation of Sph by two kinases(SphK1 and SphK2) leads to the production of S1P largely fromerythrocytes, but also from a radiation resistant source, possibly thelymphatic endothelium (Pappu, R. et al., Science, 316:295-298 (2007)).Originally thought to operate solely as an intracellular signalingmolecule, S1P was subsequently identified as a high affinity ligand forfive members of the endothelial differentiation gene (EDG) class ofG-protein coupled receptors (GPCRs) named S1P₁ or S1P1, S1P₂ or S1P2,S1P₃ or S1P3, S1P₄ or S1P4, and S1P₅ or S1P5 (formerly called EDG-1,EDG-5, EDG-3, EDG-6, and EDG-8, respectively) (Chun, J. et al.,Pharmacological Rev., 62:579-587 (2010)). The interaction of S1P withthe SIP receptors plays a fundamental physiological role in a largenumber of processes including cell proliferation, cell morphology, tumorcell invasion, angiogenesis, tumorigenesis, cytoskeletal rearrangement,vascular development, and lymphocyte trafficking (Olivera, A. et al.,Adv. Exp. Med. Biol., 716:123-142 (2011)). S1P receptors are thereforegood targets for a wide variety of therapeutic applications such astumor growth inhibition, vascular disease, and autoimmune diseases.

Among the five S1P receptors, S1P₁ has a widespread distribution. It isthe predominant family member expressed on lymphocytes and plays animportant role in lymphocyte trafficking S1P interaction with itsreceptor S1P₁ is required for the egress of immune cells from thelymphoid organs (such as thymus and lymph nodes) into the lymphaticvessels. Downregulation of the S1P₁ receptor (which can be accomplishedthrough treatment with agonists of S1P₁ via receptor internalization)disrupts lymphocyte migration and homing to various tissues. Thisresults in sequestration of the lymphocytes in lymph organs therebydecreasing the number of circulating lymphocytes that are capable ofmigration to the affected tissues. The development of an S1P₁ receptormodulating agent that suppresses lymphocyte migration to the targetsites associated with autoimmune and aberrant inflammatory processescould be efficacious in a number of autoimmune and inflammatory diseasestates.

The following applications have described compounds as S1P₁ agonists: WO03/061567 (U.S. Patent Publication No. 2005/0070506), WO 03/062248 (U.S.Pat. No. 7,351,725), WO 03/062252 (U.S. Pat. No. 7,479,504), WO03/073986 (U.S. Pat. No. 7,309,721), WO 03/105771, WO 05/058848, WO05/000833, WO 05/082089 (U.S. Patent Publication No. 2007/0203100), WO06/047195, WO 06/100633, WO 06/115188, WO 06/131336, WO 2007/024922, WO07/109330, WO 07/116866, WO 08/023783 (U.S. Patent Publication No.2008/0200535), WO 08/029370, WO 08/074820, WO 08/079382, WO 08/114157,WO 09/043889, WO 09/057079, and U.S. Pat. No. 6,069,143. Also see Haleet al., J. Med. Chem., 47:6662 (2004).

There still remains a need for compounds useful as S1P₁ agonists and yethaving selectivity over S1P₃. Further, there still remains a need forcompounds useful as S1P₁ agonists that have selectivity over S1P₃ andalso have minimal or no undesirable pulmonary effects.

Applicants have found potent compounds that have activity as S1P₁agonists. Further, applicants have found compounds that have activity asS1P₁ agonists and are selective over S1P₃. Further still, applicantshave found compounds that have activity as S1P₁ agonists, are selectiveover S1P₃, and have minimal or no undesirable pulmonary effects. Thesecompounds are provided to be useful as pharmaceuticals with desirablestability, bioavailability, therapeutic index, and toxicity values thatare important to their drugability.

SUMMARY OF THE INVENTION

The present invention provides bicyclic compounds, which are useful asmodulators of S1P₁ activity, including salts and prodrugs thereof.

The present invention also provides pharmaceutical compositionscomprising a compound of Formula (I) and/or a pharmaceuticallyacceptable salt thereof; and a pharmaceutically acceptable carrier.

The present invention also provides a method of treating a disease ordisorder associated with the activity of G protein-coupled receptorS1P₁, the method comprising administering to a mammalian patient acompound of Formula (I) and/or a pharmaceutically acceptable saltthereof.

The present invention also provides processes and intermediates formaking the compounds of Formula (I) and/or salts thereof.

The present invention also provides a compound of Formula (I) and/or apharmaceutically acceptable salt thereof, for use in therapy.

The present invention also provides the use of the compounds of Formula(I) and/or pharmaceutically acceptable salts thereof, for themanufacture of a medicament for the treatment or prophylaxis of S1P₁receptor-related conditions, such as autoimmune and vascular diseases.

The compounds of Formula (I) and compositions comprising the compoundsof Formula (I) may be used in treating, preventing, or curing variousS1P₁ related conditions. Pharmaceutical compositions comprising thesecompounds are useful in treating, preventing, or slowing the progressionof diseases or disorders in a variety of therapeutic areas, such asautoimmune and vascular diseases.

These and other features of the invention will be set forth in expandedform as the disclosure continues.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is illustrated by reference to the accompanying drawingsdescribed below.

FIG. 1 shows the experimental and the simulated PXRD patterns (CuKαλ=1.5418 Å at approximately 25° C.) of the N-1 Form of the compound ofExample 2.

FIG. 2 shows the experimental and the simulated PXRD patterns (CuKαλ=1.5418 Å at approximately 25° C.) of the monohydrate H-1 Form of themono-HCl salt of the compound of Example 2.

FIG. 3 shows the experimental and the simulated PXRD patterns (CuKαλ=1.5418 Å at approximately 25° C.) of the monohydrate H-2 Form of themono-HCl salt of the compound of Example 2.

FIG. 4 shows the experimental and the simulated PXRD patterns (CuKαλ=1.5418 Å at approximately 25° C.) of the N-3 Form of the mono-HCl saltof the compound of Example 2.

FIG. 5 shows the experimental and the simulated PXRD patterns (CuKαλ=1.5418 Å at approximately 25° C.) of the N-4 Form of the mono-HCl saltof the compound of Example 2.

FIG. 6 shows the experimental and the simulated PXRD patterns (CuKαλ=1.5418 Å at approximately 25° C.) of the monohydrate H-1 Form of thehemi-L-malic salt of the compound of Example 2.

FIG. 7 shows the experimental and the simulated PXRD patterns (CuKαλ=1.5418 Å at approximately 25° C.) of the monohydrate H-1 Form of thehemi-malonic salt of the compound of Example 2.

FIG. 8 shows the experimental and the simulated PXRD patterns (CuKαλ=1.5418 Å at approximately 25° C.) of the ⅓-hydrate H.33-1 Form of the⅔ phosphoric acid salt of the compound of Example 2.

FIG. 9 shows the simulated PXRD pattern, calculated at −70° C. (CuKαλ=1.5418 Å) of the N-1 Form of the R-(+)-mandelic acid salt of thecompound of Example 2.

DETAILED DESCRIPTION

The first aspect of the present invention provides at least one compoundof Formula (I):

and/or a salt thereof; wherein R is —OH or —OP(O)(OH)₂.

One embodiment provides compounds of Formula (I) and/or salts thereof,wherein R is —OP(O)(OH)₂. The compounds of this embodiment have thestructure of Formula (II):

Included in this embodiment are the compound of Formula (IIa) and

and the compound of Formula (IIb):

The compounds of Formula (II) and/or salts thereof, are useful asselective agonists of S1P₁.

One embodiment provides compounds of Formula (I) and/or salts thereof,wherein R is —OH. The compounds of this embodiment have the structure ofFormula (III):

Included in this embodiment are the compound of Formula (IIIa) and

and the compound of Formula (IIIb):

The compounds of Formula (III) and/or salts thereof, are useful asprodrugs of the compounds of Formula (II). The compounds of Formula(III) are activated in vivo through phosphorylation to provide thecompounds of Formula (II). The compounds of Formula (II) are active as aselective agonists of S1P₁.

One embodiment provides compounds of Formula (I) and/or salts thereof,having the structure of Formula (IV):

wherein R is —OH or —OP(O)(OH)₂. Included in this embodiment are thecompounds of Formula (IIa) and Formula (IIIa).

One embodiment provides compounds of Formula (I) and/or salts thereof,having the structure of Formula (V):

wherein R is —OH or —OP(O)(OH)₂. Included in this embodiment are thecompounds of Formula (IIb) and Formula (IIIb).

One embodiment provides the compound of Formula (IIa) and/or saltsthereof.

One embodiment provides the compound of Formula (IIb) and/or saltsthereof.

One embodiment provides the compound of Formula (IIIa) and/or saltsthereof.

One embodiment provides the compound of Formula (IIIb) and/or saltsthereof.

One embodiment provides the compound of Formula (IIa).

One embodiment provides the compound of Formula (IIb).

One embodiment provides the compound of Formula (IIIa).

One embodiment provides the compound of Formula (IIIb).

One embodiment provides one or more salts of the compound of Formula(IIa).

One embodiment provides one or more salts of the compound of Formula(IIb).

One embodiment provides one or more salts of the compound of Formula(IIIa).

One embodiment provides one or more salts of the compound of Formula(IIIb).

One embodiment provides the compound of Formula (III) as an HCl salt.

One embodiment provides the compound of Formula (IIIa) as an HCl salt.

One embodiment provides the compound of Formula (IIIb) as an HCl salt.

One embodiment provides the compound of Formula (III) as a phosphoricacid salt.

One embodiment provides the compound of Formula (IIIa) as a phosphoricacid salt.

One embodiment provides the compound of Formula (IIIb) as a phosphoricacid salt.

One embodiment provides the compound of Formula (IIIb) as an L-malicacid salt.

One embodiment provides the compound of Formula (III) as malonic acidsalt.

One embodiment provides the compound of Formula (IIIa) as malonic acidsalt.

One embodiment provides the compound of Formula (IIIb) as malonic acidsalt.

One embodiment provides the compound of Formula (IIIb) as anR-(+)-mandelic acid salt.

One embodiment provides the compound of Formula (III) as a salt selectedfrom HCl salt, phosphoric acid salt, and malonic acid salt.

One embodiment provides the compound of Formula (IIIa) as a saltselected from HCl salt, phosphoric acid salt, and malonic acid salt.

One embodiment provides the compound of Formula (IIIb) as a saltselected from HCl salt, phosphoric acid salt, L-malic acid salt, malonicacid salt, and R-(+)-mandelic acid salt.

One embodiment provides a compound selected from((1R,3R)-1-amino-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanoland((1R,3S)-1-amino-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol;and salts thereof.

One embodiment provides a compound selected from((1R,3R)-1-amino-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methyldihydrogen phosphate and((1R,3S)-1-amino-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methyldihydrogen phosphate; and salts thereof.

One embodiment provides a compound selected from((1R,3R)-1-amino-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanoland((1R,3R)-1-amino-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methyldihydrogen phosphate; and salts thereof.

One embodiment provides a compound selected from((1R,3S)-1-amino-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanoland((1R,3S)-1-amino-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methyldihydrogen phosphate; and salts thereof.

Salts and Crystal Forms of the Compound of Example 2:

TABLE 1 Salts and Crystalline Forms of Example 2 Example 2 Form freebase N-1 mono-HCl salt (monohydrate) H-1 mono-HCl salt (monohydrate) H-2mono-HCl salt N-3 mono-HCl salt N-4 hemi-L-Malic Acid salt (monohydrate)H-1 hemi-Malonic Acid salt (monohydrate) H-1 ⅔-Phosphoric Acid salt(⅓-hydrate) H.33-1 R-(+)-Mandelic Acid salt N-1Form N-1 of Example 2, Free Base

In one embodiment, the compound of Example 2

is provided as a crystalline material comprising the first crystallineform. The first crystalline form of the compound of Example 2 comprisesa neat crystalline form referred to herein as “Form N-1” or “N-1 Form”of Example 2.

In one embodiment, the N-1 Form of Example 2 is characterized by unitcell parameters approximately equal to the following:

-   -   Cell dimensions:        -   a=5.54 Å        -   b=7.37 Å        -   c=48.85 Å        -   α=90.0°        -   β=90.0°        -   γ=90.0°    -   Space group: P2₁2₁2₁    -   Molecules of Example 2/asymmetric unit: 1    -   Volume/Number of molecules in the unit cell=499 Å³    -   Density (calculated)=1.098 g/cm³,        wherein the unit cell parameters of Form N-1 of Example 2 are        measured at a temperature of about −70° C.

In another embodiment, the N-1 Form of Example 2 is characterized by asimulated powder x-ray diffraction (PXRD) pattern substantially inaccordance with the pattern shown in FIG. 1 and/or by an observed PXRDpattern substantially in accordance with the pattern shown in FIG. 1.

In yet another embodiment, the N-1 Form of Example 2 is characterized bya PXRD pattern (CuKα λ=1.5418 Å at a temperature of about 25° C.)comprising four or more, preferably five or more, 2θ values selectedfrom: 3.6±0.2, 7.2±0.2, 12.5±0.2, 14.0±0.2, 15.0±0.2, 17.5±0.2,19.4±0.2, 20.4±0.2, and 23.8±0.2, wherein the PXRD pattern of Form N-1is measured at a temperature of about 25° C.

In still yet an even further embodiment, the N-1 Form of Example 2 issubstantially pure.

In still yet another embodiment, the N-1 Form of Example 2 contains atleast about 90 wt. %, preferably at least about 95 wt. %, and morepreferably at least about 99 wt. %, based on weight of the Form N-1 ofExample 2.

In yet another embodiment, a substantially pure Form N-1 of Example 2has substantially pure phase homogeneity with less than about 10%,preferably less than about 5%, and more preferably less than about 2% ofthe total peak area of the experimentally measured PXRD pattern arisingfrom peaks that are absent from the simulated PXRD pattern. Mostpreferably, the substantially pure crystalline Form N-1 of Example 2 hassubstantially pure phase homogeneity with less than about 1% of thetotal peak area of the experimentally measured PXRD pattern arising frompeaks that are absent from the simulated PXRD pattern.

In another embodiment, the crystalline form of Example 2 consistsessentially of Form N-1. The crystalline form of this embodiment maycomprise at least about 90 wt. %, preferably at least about 95 wt. %,and more preferably at least about 99 wt. %, based on the weight of thecrystalline form, Form N-1 of Example 2.

In yet another embodiment, a pharmaceutical composition is providedcomprising Form N-1 of Example 2; and at least onepharmaceutically-acceptable carrier and/or diluent.

In still another embodiment, a pharmaceutical composition comprisessubstantially pure Form N-1 of Example 2; and at least onepharmaceutically-acceptable carrier and/or diluent.

In still an even further embodiment, a therapeutically effective amountof Form N-1 of Example 2 is combined with at least one pharmaceuticallyacceptable carrier and/or diluent to provide at least one pharmaceuticalcomposition.

HCl Salts of Example 2

In one embodiment, the Example 2 is provided as a hydrochloric acidsalt.

In one embodiment, the Example 2 is provided as a mono-hydrochloric acidsalt comprising one mole of HCl for each mole of Example 2.

In one embodiment, the mono-hydrochloric acid salt of Example 2 isprovided as crystalline material comprising one or more crystallineforms. Examples of suitable crystalline forms of the mono-hydrochloricacid salt of Example 2 include Forms H-1, H-2, N-3, and N-4.

In one embodiment, the mono-hydrochloric acid salt of Example 2 isprovided as a monohydrate.

In one embodiment, the monohydrate mono-hydrochloric acid salt ofExample 2 is provided in as crystalline material comprising one or morecrystalline forms. Examples of suitable crystalline forms of themonohydrate mono-hydrochloric acid salt of Example 2 include Forms H-1and H-2.

In one embodiment, the monohydrate mono-hydrochloric acid salt ofExample 2 is provided in a second crystalline form referred to herein as“Form H-1” or “H-1 Form” of Example 2, HCl salt. The H-1 Form of Example2, HCl salt comprises one molecule of water and one molecule of HCl foreach molecule of Example 2.

In one embodiment, the H-1 Form of the mono-hydrochloric acid salt ofExample 2 is characterized by unit cell parameters approximately equalto the following:

-   -   Cell dimensions:        -   a=6.30 Å        -   b=6.42 Å        -   c=55.28 Å        -   α=90.0°        -   β=90.0°        -   γ=90.0°    -   Space group: P2₁2₁2₁    -   Molecules of Example 2/asymmetric unit: 1    -   Volume/number of molecules in the unit cell=560 Å³    -   Density (calculated)=1.14 g/cm³,        wherein the unit cell parameters of Form H-1 of the        mono-hydrochloric acid salt of Example 2 are measured at a        temperature of about −70° C.

In another embodiment, the H-1 Form of the mono-hydrochloric acid saltof Example 2 is characterized by a simulated powder x-ray diffraction(PXRD) pattern substantially in accordance with the pattern shown inFIG. 2 and/or by an observed PXRD pattern substantially in accordancewith the pattern shown in FIG. 2.

In yet another embodiment, the monohydrate H-1 form of the mono-HCl saltof Example 2 is characterized by a PXRD pattern (CuKα λ=1.5418 Å at atemperature of about 25° C.) comprising four or more, preferably five ormore, 2θ values selected from: 3.2±0.2, 6.4±0.2, 9.6±0.2, 13.9±0.2,14.6±0.2, 17.0±0.2, 19.0±0.2, 20.1±0.2, 21.3±0.2, 21.9±0.2, and24.5±0.2, wherein the PXRD pattern is measured at a temperature of about25° C.

In still yet an even further embodiment, the H-1 Form of themono-hydrochloric acid salt of Example 2 is substantially pure.

In still yet another embodiment, the H-1 Form of the mono-hydrochloricacid salt of Example 2 contains at least about 90 wt. %, preferably atleast about 95 wt. %, and more preferably at least about 99 wt. %, basedon weight of the second crystalline form, Form H-1 of Example 2, HClsalt.

In yet another embodiment, a substantially pure second crystalline formhas substantially pure phase homogeneity with less than about 10%,preferably less than about 5%, and more preferably less than about 2% ofthe total peak area of the experimentally measured PXRD pattern arisingfrom peaks that are absent from the simulated PXRD pattern. Mostpreferably, a substantially pure second crystalline form hassubstantially pure phase homogeneity with less than about 1% of thetotal peak area of the experimentally measured PXRD pattern arising frompeaks that are absent from the simulated PXRD pattern.

In another embodiment, the second crystalline form of the mono-HCl saltof Example 2 consists essentially of Form H-1. The second crystallineform of this embodiment may comprise at least about 90 wt. %, preferablyat least about 95 wt. %, and more preferably at least about 99 wt. %,based on the weight of the second crystalline form, Form H-1.

In one embodiment, the monohydrate mono-hydrochloric acid salt ofExample 2 is provided in a third crystalline form referred to herein as“Form H-2” or “H-2 Form” of Example 2, HCl salt. The H-2 Form of Example2, HCl salt comprises one molecule of water and one molecule of HCl foreach molecule of Example 2.

In one embodiment, the H-2 Form of the mono-hydrochloric acid salt ofExample 2 is characterized by unit cell parameters approximately equalto the following:

-   -   Cell dimensions:        -   a=6.30 Å        -   b=6.43 Å        -   c=27.88 Å        -   α=90.0°        -   β=96.0°        -   γ=90.0°    -   Space group: P2₁    -   Molecules of Example 2/asymmetric unit: 1    -   Volume/number of molecules in the unit cell=562 Å³    -   Density (calculated)=1.135 g/cm³,        wherein the unit cell parameters of the H-2 Form of Example 2,        HCl salt are measured at a temperature of about −70° C.

In another embodiment, the H-2 Form of Example 2, HCl salt ischaracterized by a simulated powder x-ray diffraction (PXRD) patternsubstantially in accordance with the pattern shown in FIG. 3 and/or byan observed PXRD pattern substantially in accordance with the patternshown in FIG. 3.

In yet another embodiment, the monohydrate H-2 form of the mono-HCl saltof Example 2 is characterized by a PXRD pattern (CuKα λ=1.5418 Å at atemperature of about 25° C.) comprising four or more, preferably five ormore, 2θ values selected from: 3.2±0.2, 6.4±0.2, 9.6±0.2, 14.1±0.2,15.2±0.2, 16.8±0.2, 18.8±0.2, 20.2±0.2, 21.3±0.2, 22.6±0.2, and26.6±0.2, wherein the PXRD pattern is measured at a temperature of about25° C.

In still yet an even further embodiment, the H-2 Form of Example 2, HClsalt is substantially pure.

In still yet another embodiment, the H-2 Form of Example 2, HCl saltcontains at least about 90 wt. %, preferably at least about 95 wt. %,and more preferably at least about 99 wt. %, based on weight of thethird crystalline form.

In yet another embodiment, a substantially pure H-2 Form of Example 2,HCl salt has substantially pure phase homogeneity with less than about10%, preferably less than about 5%, and more preferably less than about2% of the total peak area of the experimentally measured PXRD patternarising from peaks that are absent from the simulated PXRD pattern. Mostpreferably, the substantially crystalline H-2 Form of Example 2, HClsalt has substantially pure phase homogeneity with less than about 1% ofthe total peak area of the experimentally measured PXRD pattern arisingfrom peaks that are absent from the simulated PXRD pattern.

In another embodiment, the third crystalline form of the compound ofExample 2 consists essentially of H-2 Form of Example 2, HCl salt. Thethird crystalline form of this embodiment may comprise at least about 90wt. %, preferably at least about 95 wt. %, and more preferably at leastabout 99 wt. %, based on the weight of the third crystalline form.

In one embodiment, the mono-hydrochloric acid salt of Example 2 isprovided as neat crystalline material comprising one or more crystallineforms. Examples of suitable crystalline forms of the neatmono-hydrochloric acid salt of Example 2 include Forms N-3 and N-4.

In one embodiment, the neat mono-hydrochloric acid salt of Example 2 isprovided in a fourth crystalline form referred to herein as “Form N-3”or “N-3 Form” of Example 2, HCl salt. The N-3 Form of Example 2, HClsalt comprises one molecule of HCl for each molecule of Example 2.

In one embodiment, the N-3 Form of the HCl salt of Example 2 ischaracterized by unit cell parameters approximately equal to thefollowing:

-   -   Cell dimensions:        -   a=5.98 Å        -   b=7.24 Å        -   c=25.74 Å        -   α=90.0°        -   β=91.7°        -   γ=90.0°    -   Space group: P2₁    -   Molecules of Example 2/asymmetric unit: 1    -   Volume/number of molecules in the unit cell=556 Å³    -   Density (calculated)=1.092 g/cm³,        wherein the unit cell parameters of Form N-3 of the HCl salt of        Example 2 are measured at a temperature of about 27° C.

In one embodiment, the N-3 Form of the HCl salt of Example 2 ischaracterized by unit cell parameters approximately equal to thefollowing:

-   -   Cell dimensions:        -   a=5.93 Å        -   b=7.20 Å        -   c=25.24 Å        -   a=90.0°        -   β=90.2°        -   γ=90.0°    -   Space group: P2₁    -   Molecules of Example 2/asymmetric unit: 1    -   Volume/number of molecules in the unit cell=538 Å³    -   Density (calculated)=1.128 g/cm³,        wherein the unit cell parameters of Form N-3 of the HCl salt of        Example 2 are measured at a temperature of about −70° C.

In another embodiment, the N-3 Form of Example 2, HCl salt ischaracterized by a simulated powder x-ray diffraction (PXRD) patternsubstantially in accordance with the pattern shown in FIG. 4 and/or byan observed PXRD pattern substantially in accordance with the patternshown in FIG. 4.

In yet another embodiment, the N-3 form of the mono-HCl salt of Example2 is characterized by a PXRD pattern (CuKα λ=1.5418 Å at a temperatureof about 25° C.) comprising four or more, preferably five or more, 2θvalues selected from: 3.4±0.2, 6.9±0.2, 10.4±0.2, 12.7±0.2, 14.0±0.2,16.1±0.2, 19.7±0.2, 20.6±0.2, 22.1±0.2, and 24.0±0.2, wherein the PXRDpattern is measured at a temperature of about 25° C.

In still yet an even further embodiment, the N-3 Form of the HCl salt ofExample 2 is substantially pure.

In still yet another embodiment, the N-3 Form of the HCl salt of Example2 contains at least about 90 wt. %, preferably at least about 95 wt. %,and more preferably at least about 99 wt. %, based on weight of thefourth crystalline form, Form N-3.

In yet another embodiment, a substantially pure Form N-3 of the HCl saltof Example 2 has substantially pure phase homogeneity with less thanabout 10%, preferably less than about 5%, and more preferably less thanabout 2% of the total peak area of the experimentally measured PXRDpattern arising from peaks that are absent from the simulated PXRDpattern. Most preferably, the substantially crystalline Form N-3 of theHCl salt of Example 2 has substantially pure phase homogeneity with lessthan about 1% of the total peak area of the experimentally measured PXRDpattern arising from peaks that are absent from the simulated PXRDpattern.

In still yet an even further embodiment, the Form N-3 of the HCl salt ofExample 2 is substantially pure.

In another embodiment, the crystalline form of Example 2 consistsessentially of Form N-3. The crystalline form of this embodiment maycomprise at least about 90 wt. %, preferably at least about 95 wt. %,and more preferably at least about 99 wt. %, based on the weight of thecrystalline form, Form N-3 of Example 2.

In yet another embodiment, a pharmaceutical composition is providedcomprising Form N-3 of Example 2; and at least onepharmaceutically-acceptable carrier and/or diluent.

In still another embodiment, a pharmaceutical composition comprisessubstantially pure Form N-3 of Example 2; and at least onepharmaceutically-acceptable carrier and/or diluent.

In still an even further embodiment, a therapeutically effective amountof Form N-3 of Example 2 is combined with at least one pharmaceuticallyacceptable carrier and/or diluent to provide at least one pharmaceuticalcomposition.

In another embodiment, the fourth crystalline form of the HCl salt ofExample 2 consists essentially of Form N-3. The fourth crystalline formof this embodiment may comprise at least about 90 wt. %, preferably atleast about 95 wt. %, and more preferably at least about 99 wt. %, basedon the weight of the fourth crystalline form, Form N-3.

In one embodiment, the neat mono-hydrochloric acid salt of Example 2 isprovided in a fifth crystalline form referred to herein as “Form N-4” or“N-4 Form” of Example 2, HCl salt. The N-4 Form of Example 2, HCl saltcomprises one molecule of HCl for each molecule of Example 2.

In one embodiment, the N-4 Form of the HCl salt of Example 2 ischaracterized by unit cell parameters approximately equal to thefollowing:

-   -   Cell dimensions:        -   a=5.95 Å        -   b=7.27 Å        -   c=51.60 Å        -   α=90.0°        -   β=90.0°        -   γ=90.0°    -   Space group: P2₁2₁2₁    -   Molecules of Example 2/asymmetric unit: 1    -   Volume/number of molecules in the unit cell=558 Å³    -   Density (calculated)=1.088 g/cm³,        wherein the unit cell parameters of Form N-4 of Example 2, HCl        salt are measured at a temperature of about 27° C.

In one embodiment, the N-4 Form of the HCl salt of Example 2 ischaracterized by unit cell parameters approximately equal to thefollowing:

-   -   Cell dimensions:        -   a=5.92 Å        -   b=7.25 Å        -   c=50.61 Å        -   α=90.0°        -   β=90.0°        -   γ=90.0°    -   Space group: P2₁2₁2₁    -   Molecules of Example 2/asymmetric unit: 1    -   Volume/number of molecules in the unit cell=543 Å³    -   Density (calculated)=1.119 g/cm³,        wherein the unit cell parameters of Form N-4 of Example 2, HCl        salt are measured at a temperature of about −70° C.

In another embodiment, the N-4 Form of Example 2, HCl salt ischaracterized by a simulated powder x-ray diffraction (PXRD) patternsubstantially in accordance with the pattern shown in FIG. 5 and/or byan observed PXRD pattern substantially in accordance with the patternshown in FIG. 5.

In yet another embodiment, the N-4 form of the mono-HCl salt of Example2 is characterized by a PXRD pattern (CuKα λ=1.5418 Å at a temperatureof about 25° C.) comprising four or more, preferably five or more, 2θvalues selected from: 3.4±0.2, 6.9±0.2, 10.3±0.2, 12.7±0.2, 13.3±0.2,15.0±0.2, 19.7±0.2, 20.4±0.2, 21.2±0.2, 22.9±0.2, and 24.9±0.2, whereinthe PXRD pattern is measured at a temperature of about 25° C.

In still yet an even further embodiment, the N-4 Form of the HCl salt ofExample 2 is substantially pure.

In still yet another embodiment, the N-4 Form of the HCl salt of Example2 contains at least about 90 wt. %, preferably at least about 95 wt. %,and more preferably at least about 99 wt. %, based on weight of thefourth crystalline form, Form N-4.

In yet another embodiment, a substantially pure Form N-4 of the HCl saltof Example 2 has substantially pure phase homogeneity with less thanabout 10%, preferably less than about 5%, and more preferably less thanabout 2% of the total peak area of the experimentally measured PXRDpattern arising from peaks that are absent from the simulated PXRDpattern. Most preferably, the substantially crystalline Form N-4 of theHCl salt of Example 2 has substantially pure phase homogeneity with lessthan about 1% of the total peak area of the experimentally measured PXRDpattern arising from peaks that are absent from the simulated PXRDpattern.

In still yet an even further embodiment, the Form N-4 of the HCl salt ofExample 2 is substantially pure.

In another embodiment, the crystalline form of Example 2 consistsessentially of Form N-4. The crystalline form of this embodiment maycomprise at least about 90 wt. %, preferably at least about 95 wt. %,and more preferably at least about 99 wt. %, based on the weight of thecrystalline form, Form N-4 of Example 2.

In yet another embodiment, a pharmaceutical composition is providedcomprising Form N-4 of Example 2; and at least onepharmaceutically-acceptable carrier and/or diluent.

In still another embodiment, a pharmaceutical composition comprisessubstantially pure Form N-4 of Example 2; and at least onepharmaceutically-acceptable carrier and/or diluent.

In still an even further embodiment, a therapeutically effective amountof Form N-4 of Example 2 is combined with at least one pharmaceuticallyacceptable carrier and/or diluent to provide at least one pharmaceuticalcomposition.

In one embodiment, a composition is provided comprising Form N-3, FormN-4, or a mixture thereof of the neat mono-hydrochloric acid salt ofExample 2.

L-Malic Acid Salt

In one embodiment, the Example 2 is provided as an L-malic acid salt.

In one embodiment, the Example 2 is provided as a hemi-L-malic acid saltcomprising 0.5 mole of L-malic acid for each mole of Example 2.

In one embodiment, the Example 2 is provided as a monohydratehemi-L-malic acid salt comprising one mole of water and 0.5 mole ofL-malic acid for each mole of Example 2.

In one embodiment, the monohydrate hemi-L-malic acid salt of Example 2is provided in a sixth crystalline form referred to herein as “Form H-1”or “H-1 Form” of Example 2, hemi-L-malic acid salt. The H-1 Form ofExample 2, hemi-L-malic acid salt comprises one molecule of water and0.5 molecule of L-malic acid for each molecule of Example 2.

In one embodiment, the H-1 Form of the hemi-L-malic acid salt of Example2 is characterized by unit cell parameters approximately equal to thefollowing:

-   -   Cell dimensions:        -   a=6.13 Å        -   b=13.83 Å        -   c=28.75 Å        -   α=103.4°        -   β=94.0°        -   γ=92.6°    -   Space group: P1    -   Molecules of Example 2/asymmetric unit: 4    -   Volume/number of molecules in the unit cell=591 Å³    -   Density (calculated)=1.165 g/cm³,        wherein the unit cell parameters of Form H-1 of the hemi-L-malic        acid salt of Example 2 are measured at a temperature of about        −70° C.

In another embodiment, the H-1 form of the hemi-L-malic acid salt ofExample 2 is characterized by a simulated powder x-ray diffraction(PXRD) pattern substantially in accordance with the pattern shown inFIG. 6 and/or by an observed PXRD pattern substantially in accordancewith the pattern shown in FIG. 6.

In yet another embodiment, the H-1 form of the hemi-L-malic acid salt ofExample 2 is characterized by a PXRD pattern (CuKα λ=1.5418 Å at atemperature of about 25° C.) comprising four or more, preferably five ormore, 2θ values selected from: 3.2±0.2, 6.3±0.2, 9.5±0.2, 12.8±0.2,14.3±0.2, 18.0±0.2, 22.4±0.2, and 24.8±0.2, wherein the PXRD pattern ismeasured at a temperature of about 25° C.

Malonic Acid Salt

In one embodiment, the Example 2 is provided as a malonic acid salt.

In one embodiment, the Example 2 is provided as a hemi-malonic acid saltcomprising 0.5 mole of malonic acid for each mole of Example 2.

In one embodiment, the Example 2 is provided as a monohydratehemi-malonic acid salt comprising one mole of water and 0.5 mole ofmalonic acid for each mole of Example 2.

In one embodiment, the monohydrate hemi-malonic acid salt of Example 2is provided in a seventh crystalline form referred to herein as “FormH-1” or “H-1 Form” of Example 2, hemi-malonic acid salt. The H-1 Form ofExample 2, hemi malonic acid salt comprises one molecule of water and0.5 molecule of malonic acid for each molecule of Example 2.

In one embodiment, the H-1 Form of the hemi-malonic acid salt of Example2 is characterized by unit cell parameters approximately equal to thefollowing:

-   -   Cell dimensions:        -   a=14.73 Å        -   b=6.27 Å        -   c=25.36 Å        -   α=90.0°        -   β=93.8°        -   γ=90.0°    -   Space group: P2₁    -   Molecules of Example 2/asymmetric unit: 2    -   Volume/number of molecules in the unit cell=584 Å³    -   Density (calculated)=1.137 g/cm³,        wherein the unit cell parameters of Form H-1 are measured at a        temperature of about −70° C.

In another embodiment, the H-1 form of the hemi-malonic acid salt ofExample 2 is characterized by a simulated powder x-ray diffraction(PXRD) pattern substantially in accordance with the pattern shown inFIG. 7 and/or by an observed PXRD pattern substantially in accordancewith the pattern shown in FIG. 7.

In yet another embodiment, the H-1 form of the hemi-malonic acid salt ofExample 2 is characterized by a PXRD pattern (CuKα λ=1.5418 Å at atemperature of about 25° C.) comprising four or more, preferably five ormore, 2θ values selected from: 3.5±0.2, 7.1±0.2, 12.3±0.2, 13.5±0.2,15.5±0.2, 17.6±0.2, 19.1±0.2, 20.2±0.2, 20.6±0.2, 21.7±0.2, and23.8±0.2, wherein the PXRD pattern is measured at a temperature of about25° C.

Phosphoric Acid Salt

In one embodiment, the Example 2 is provided as a phosphoric acid salt.

In one embodiment, the Example 2 is provided as a ⅓-hydrate phosphoricacid salt.

In one embodiment, the Example 2 is provided as a phosphoric acid saltcomprising 0.67 mole of phosphoric acid for each mole of Example 2.

In one embodiment, the Example 2 is provided as a ⅓-hydrate phosphoricacid salt comprising 0.33 mole of water and 0.67 mole of phosphoric acidfor each mole of Example 2.

In one embodiment, the ⅓-hydrate phosphoric acid salt of Example 2 isprovided in an eighth crystalline form referred to herein as “FormH.33-1” or “H.33-1 Form” of Example 2, phosphoric acid salt. The H.33-1Form of Example 2, phosphoric acid salt comprises 0.33 molecule of waterand 0.67 molecule of phosphoric acid for each molecule of Example 2.

In one embodiment, the H.33-1 Form is characterized by unit cellparameters approximately equal to the following:

-   -   Cell dimensions:        -   a=59.12 Å        -   b=6.89 Å        -   c=16.62 Å        -   α=90.0°        -   β=94.7°        -   γ=90.0°    -   Space group: C2    -   Molecules of Example 2/asymmetric unit: 3    -   Volume/number of molecules in the unit cell=563 Å³    -   Density (calculated)=1.181 g/cm³,        wherein the unit cell parameters of Form H.33-1 are measured at        a temperature of about −70° C.

In another embodiment, the H.33-1 form of the ⅓-phosphoric acid salt ofExample 2 is characterized by a simulated powder x-ray diffraction(PXRD) pattern substantially in accordance with the pattern shown inFIG. 8 and/or by an observed PXRD pattern substantially in accordancewith the pattern shown in FIG. 8.

In yet another embodiment, the H.33-1 form of the ⅓-phosphoric acid saltof Example 2 is characterized by a PXRD pattern (CuKα λ=1.5418 Å at atemperature of about 25° C.) comprising four or more, preferably five ormore, 2θ values selected from: 2.9±0.2, 5.9±0.2, 8.8±0.2, 13.9±0.2,15.8±0.2, 16.7±0.2, 17.4±0.2, 18.4±0.2, 19.4±0.2, and 20.4±0.2, whereinthe PXRD pattern is measured at a temperature of about 25° C.

R-(+)-Mandelic Acid Salt

In one embodiment, the Example 2 is provided as an R-(+)-mandelic acidsalt.

In one embodiment, the Example 2 is provided as a monohydrateR-(+)-mandelic acid.

In one embodiment, the Example 2 is provided as an R-(+)-mandelic acidsalt comprising one mole of R-(+)-mandelic acid for each mole of Example2.

In one embodiment, the Example 2 is provided as a monohydrateR-(+)-mandelic acid salt comprising one mole of water and one mole ofR-(+)-mandelic acid for each mole of Example 2.

In one embodiment, the monohydrate R-(+)-mandelic acid salt of Example 2is provided in an ninth crystalline form referred to herein as “FormN-1” or “N-1 Form” of Example 2, R-(+)-mandelic acid salt. The N-1 Formof Example 2, R-(+)-mandelic acid salt comprises one molecule of waterand one molecule of R-(+)-mandelic acid for each molecule of Example 2.

In one embodiment, the N-1 Form of Example 2, R-(+)-mandelic acid saltis characterized by unit cell parameters approximately equal to thefollowing:

-   -   Cell dimensions:        -   a=6.31 Å        -   b=10.03 Å        -   c=21.79 Å        -   a=98.2°        -   β=91.3°        -   γ=91.7°    -   Space group: P1    -   Molecules of Example 2/asymmetric unit: 2    -   Volume/number of molecules in the unit cell=683 Å³    -   Density (calculated)=1.171 g/cm³,        wherein the unit cell parameters of Form N-1 of Example 2,        R-(+)-mandelic acid salt are measured at a temperature of about        −70° C.

In another embodiment, the N-1 Form of Example 2, R-(+)-mandelic acidsalt is characterized by a simulated powder x-ray diffraction (PXRD)pattern substantially in accordance with the pattern shown in FIG. 9.

In still yet an even further embodiment, the N-1 Form of Example 2,R-(+)-mandelic acid salt is substantially pure.

In still yet another embodiment, the N-1 Form of Example 2,R-(+)-mandelic acid salt contains at least about 90 wt. %, preferably atleast about 95 wt. %, and more preferably at least about 99 wt. %, basedon weight of the Form N-1 of Example 2, R-(+)-mandelic acid salt.

In yet another embodiment, a substantially pure Form N-1 of Example 2,R-(+)-mandelic acid salt has substantially pure phase homogeneity withless than about 10%, preferably less than about 5%, and more preferablyless than about 2% of the total peak area of the experimentally measuredPXRD pattern arising from peaks that are absent from the simulated PXRDpattern. Most preferably, the substantially pure crystalline Form N-1 ofExample 2, R-(+)-mandelic acid salt has substantially pure phasehomogeneity with less than about 1% of the total peak area of theexperimentally measured PXRD pattern arising from peaks that are absentfrom the simulated PXRD pattern.

In another embodiment, the crystalline form of Example 2, R-(+)-mandelicacid salt consists essentially of Form N-1. The crystalline form of thisembodiment may comprise at least about 90 wt. %, preferably at leastabout 95 wt. %, and more preferably at least about 99 wt. %, based onthe weight of the crystalline form, Form N-1 of Example 2,R-(+)-mandelic acid salt.

In yet another embodiment, a pharmaceutical composition is providedcomprising Form N-1 of Example 2, R-(+)-mandelic acid salt; and at leastone pharmaceutically-acceptable carrier and/or diluent.

In still another embodiment, a pharmaceutical composition comprisessubstantially pure Form N-1 of Example 2, R-(+)-mandelic acid salt; andat least one pharmaceutically-acceptable carrier and/or diluent.

In still an even further embodiment, a therapeutically effective amountof Form N-1 of Example 2, R-(+)-mandelic acid salt is combined with atleast one pharmaceutically acceptable carrier and/or diluent to provideat least one pharmaceutical composition.

The present invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof. Thisinvention encompasses all combinations of the aspects and/or embodimentsof the invention noted herein. It is understood that any and allembodiments of the present invention may be taken in conjunction withany other embodiment or embodiments to describe additional embodiments.It is also to be understood that each individual element of theembodiments is meant to be combined with any and all other elements fromany embodiment to describe an additional embodiment.

Definitions

The features and advantages of the invention may be more readilyunderstood by those of ordinary skill in the art upon reading thefollowing detailed description. It is to be appreciated that certainfeatures of the invention that are, for clarity reasons, described aboveand below in the context of separate embodiments, may also be combinedto form a single embodiment. Conversely, various features of theinvention that are, for brevity reasons, described in the context of asingle embodiment, may also be combined so as to form sub-combinationsthereof. Embodiments identified herein as exemplary or preferred areintended to be illustrative and not limiting.

Unless specifically stated otherwise herein, references made in thesingular may also include the plural. For example, “a” and “an” mayrefer to either one, or one or more.

As used herein, the phase “compounds and/or salts thereof” refers to atleast one compound, at least one salt of the compounds, or a combinationthereof. For example, compounds of Formula (I) and/or salts thereofincludes a compound of Formula (I); two compounds of Formula (I); a saltof a compound of Formula (I); a compound of Formula (I) and one or moresalts of the compound of Formula (I); and two or more salts of acompound of Formula (I).

Unless otherwise indicated, any atom with unsatisfied valences isassumed to have hydrogen atoms sufficient to satisfy the valences.

Listed below are definitions of various terms used to describe thepresent invention. These definitions apply to the terms as they are usedthroughout the specification (unless they are otherwise limited inspecific instances) either individually or as part of a larger group.The definitions set forth herein take precedence over definitions setforth in any patent, patent application, and/or patent applicationpublication incorporated herein by reference.

Throughout the specification, groups and substituents thereof may bechosen by one skilled in the field to provide stable moieties andcompounds.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The compounds of Formula (I) can form salts which are also within thescope of this invention. Unless otherwise indicated, reference to aninventive compound is understood to include reference to one or moresalts thereof. The term “salt(s)” denotes acidic and/or basic saltsformed with inorganic and/or organic acids and bases. In addition, theterm “salt(s)” may include zwitterions (inner salts), e.g., when acompound of Formula (I) contains both a basic moiety, such as an amineor a pyridine or imidazole ring, and an acidic moiety, such as acarboxylic acid. Pharmaceutically acceptable (i.e., non-toxic,physiologically acceptable) salts are preferred, such as, for example,acceptable metal and amine salts in which the cation does not contributesignificantly to the toxicity or biological activity of the salt.However, other salts may be useful, e.g., in isolation or purificationsteps which may be employed during preparation, and thus, arecontemplated within the scope of the invention. Salts of the compoundsof the formula (I) may be formed, for example, by reacting a compound ofthe Formula (I) with an amount of acid or base, such as an equivalentamount, in a medium such as one in which the salt precipitates or in anaqueous medium followed by lyophilization.

Exemplary acid addition salts include acetates (such as those formedwith acetic acid or trihaloacetic acid, for example, trifluoroaceticacid), adipates, alginates, ascorbates, aspartates, benzoates,benzenesulfonates, bisulfates, borates, butyrates, citrates,camphorates, camphorsulfonates, cyclopentanepropionates, digluconates,dodecylsulfates, ethanesulfonates, fumarates, glucoheptanoates,glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides(formed with hydrochloric acid), hydrobromides (formed with hydrogenbromide), hydroiodides, maleates (formed with maleic acid),2-hydroxyethanesulfonates, lactates, methanesulfonates (formed withmethanesulfonic acid), 2-naphthalenesulfonates, nicotinates, nitrates,oxalates, pectinates, persulfates, 3-phenylpropionates, phosphates,picrates, pivalates, propionates, salicylates, succinates, sulfates(such as those formed with sulfuric acid), sulfonates (such as thosementioned herein), tartrates, thiocyanates, toluenesulfonates such astosylates, undecanoates, and the like.

Exemplary basic salts include ammonium salts, alkali metal salts such assodium, lithium, and potassium salts; alkaline earth metal salts such ascalcium and magnesium salts; barium, zinc, and aluminum salts; saltswith organic bases (for example, organic amines) such as trialkylaminessuch as triethylamine, procaine, dibenzylamine,N-benzyl-β-phenethylamine, 1-ephenamine, N,N′-dibenzylethylene-diamine,dehydroabietylamine, N-ethylpiperidine, benzylamine, dicyclohexylamineor similar pharmaceutically acceptable amines and salts with amino acidssuch as arginine, lysine and the like. Basic nitrogen-containing groupsmay be quaternized with agents such as lower alkyl halides (e.g.,methyl, ethyl, propyl, and butyl chlorides, bromides and iodides),dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl, and diamylsulfates), long chain halides (e.g., decyl, lauryl, myristyl and stearylchlorides, bromides and iodides), aralkyl halides (e.g., benzyl andphenethyl bromides), and others. Preferred salts includemonohydrochloride, hydrogensulfate, methanesulfonate, phosphate ornitrate salts.

The compounds of Formula (I) can be provided as amorphous solids orcrystalline solids. Lyophilization can be employed to provide thecompounds of Formula (I) as a solid.

It should further be understood that solvates (e.g., hydrates) of theCompounds of Formula (I) are also within the scope of the presentinvention. The term “solvate” means a physical association of a compoundof Formula (I) with one or more solvent molecules, whether organic orinorganic. This physical association includes hydrogen bonding. Incertain instances the solvate will be capable of isolation, for examplewhen one or more solvent molecules are incorporated in the crystallattice of the crystalline solid. “Solvate” encompasses bothsolution-phase and isolable solvates. Exemplary solvates includehydrates, ethanolates, methanolates, isopropanolates, acetonitrilesolvates, and ethyl acetate solvates. Methods of solvation are known inthe art.

Various forms of prodrugs are well known in the art and are describedin:

-   a) Wermuth, C. G. et al., The Practice of Medicinal Chemistry,    Chapter 31, Academic Press (1996);-   b) Bundgaard, H. ed., Design of Prodrugs, Elsevier (1985);-   c) Bundgaard, H., Chapter 5, “Design and Application of Prodrugs”,    Krogsgaard-Larsen, P. et al., eds., A Textbook of Drug Design and    Development, pp. 113-191, Harwood Academic Publishers (1991); and-   d) Testa, B. et al., Hydrolysis in Drug and Prodrug Metabolism,    Wiley-VCH (2003).

In addition, compounds of Formula (I), subsequent to their preparation,can be isolated and purified to obtain a composition containing anamount by weight equal to or greater than 99% of a compound of Formula(I) (“substantially pure”), which is then used or formulated asdescribed herein. Such “substantially pure” compounds of Formula (I) arealso contemplated herein as part of the present invention.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent. The present invention is intended toembody stable compounds.

“Therapeutically effective amount” is intended to include an amount of acompound of the present invention alone or an amount of the combinationof compounds claimed or an amount of a compound of the present inventionin combination with other active ingredients effective to act as anagonist to S1P₁, or effective to treat or prevent autoimmune and/orinflammatory disease states, such as multiple sclerosis and rheumatoidarthritis.

As used herein, “treating” or “treatment” cover the treatment of adisease-state in a mammal, particularly in a human, and include: (a)preventing the disease-state from occurring in a mammal, in particular,when such mammal is predisposed to the disease-state but has not yetbeen diagnosed as having it; (b) inhibiting the disease-state, i.e.,arresting its development; and/or (c) relieving the disease-state, i.e.,causing regression of the disease state.

The compounds of the present invention are intended to include allisotopes of atoms occurring in the present compounds. Isotopes includethose atoms having the same atomic number but different mass numbers. Byway of general example and without limitation, isotopes of hydrogeninclude deuterium (D) and tritium (T). Isotopes of carbon include ¹³Cand ¹⁴C. Isotopically-labeled compounds of the invention can generallybe prepared by conventional techniques known to those skilled in the artor by processes analogous to those described herein, using anappropriate isotopically-labeled reagent in place of the non-labeledreagent otherwise employed.

Compounds in accordance with Formula (I) and/or pharmaceuticallyacceptable salts thereof can be administered by any means suitable forthe condition to be treated, which can depend on the need forsite-specific treatment or quantity of Formula (I) compound to bedelivered.

Also embraced within this invention is a class of pharmaceuticalcompositions comprising a compound of Formula (I) and/orpharmaceutically acceptable salts thereof; and one or more non-toxic,pharmaceutically-acceptable carriers and/or diluents and/or adjuvants(collectively referred to herein as “carrier” materials) and, ifdesired, other active ingredients. The compounds of Formula (I) may beadministered by any suitable route, preferably in the form of apharmaceutical composition adapted to such a route, and in a doseeffective for the treatment intended. The compounds and compositions ofthe present invention may, for example, be administered orally,mucosally, or parentally including intravascularly, intravenously,intraperitoneally, subcutaneously, intramuscularly, and intrasternallyin dosage unit formulations containing conventional pharmaceuticallyacceptable carriers, adjuvants, and vehicles. For example, thepharmaceutical carrier may contain a mixture of mannitol or lactose andmicrocrystalline cellulose. The mixture may contain additionalcomponents such as a lubricating agent, e.g., magnesium stearate and adisintegrating agent such as crospovidone. The carrier mixture may befilled into a gelatin capsule or compressed as a tablet. Thepharmaceutical composition may be administered as an oral dosage form oran infusion, for example.

For oral administration, the pharmaceutical composition may be in theform of, for example, a tablet, capsule, liquid capsule, suspension, orliquid. The pharmaceutical composition is preferably made in the form ofa dosage unit containing a particular amount of the active ingredient.For example, the pharmaceutical composition may be provided as a tabletor capsule comprising an amount of active ingredient in the range offrom about 0.1 to 1000 mg, preferably from about 0.25 to 250 mg, andmore preferably from about 0.5 to 100 mg. A suitable daily dose for ahuman or other mammal may vary widely depending on the condition of thepatient and other factors, but, can be determined using routine methods.

Any pharmaceutical composition contemplated herein can, for example, bedelivered orally via any acceptable and suitable oral preparations.Exemplary oral preparations, include, but are not limited to, forexample, tablets, troches, lozenges, aqueous and oily suspensions,dispersible powders or granules, emulsions, hard and soft capsules,liquid capsules, syrups, and elixirs. Pharmaceutical compositionsintended for oral administration can be prepared according to anymethods known in the art for manufacturing pharmaceutical compositionsintended for oral administration. In order to provide pharmaceuticallypalatable preparations, a pharmaceutical composition in accordance withthe invention can contain at least one agent selected from sweeteningagents, flavoring agents, coloring agents, demulcents, antioxidants, andpreserving agents.

A tablet can, for example, be prepared by admixing at least one compoundof Formula (I) and/or at least one pharmaceutically acceptable saltthereof with at least one non-toxic pharmaceutically acceptableexcipient suitable for the manufacture of tablets. Exemplary excipientsinclude, but are not limited to, for example, inert diluents, such as,for example, calcium carbonate, sodium carbonate, lactose, calciumphosphate, and sodium phosphate; granulating and disintegrating agents,such as, for example, microcrystalline cellulose, sodium croscarmellose,corn starch, and alginic acid; binding agents, such as, for example,starch, gelatin, polyvinyl-pyrrolidone, and acacia; and lubricatingagents, such as, for example, magnesium stearate, stearic acid, andtalc. Additionally, a tablet can either be uncoated, or coated by knowntechniques to either mask the bad taste of an unpleasant tasting drug,or delay disintegration and absorption of the active ingredient in thegastrointestinal tract thereby sustaining the effects of the activeingredient for a longer period. Exemplary water soluble taste maskingmaterials, include, but are not limited to,hydroxypropyl-methylcellulose and hydroxypropyl-cellulose. Exemplarytime delay materials, include, but are not limited to, ethyl celluloseand cellulose acetate butyrate.

Hard gelatin capsules can, for example, be prepared by mixing at leastone compound of Formula (I) and/or at least one salt thereof with atleast one inert solid diluent, such as, for example, calcium carbonate;calcium phosphate; and kaolin.

Soft gelatin capsules can, for example, be prepared by mixing at leastone compound of Formula (I) and/or at least one pharmaceuticallyacceptable salt thereof with at least one water soluble carrier, suchas, for example, polyethylene glycol; and at least one oil medium, suchas, for example, peanut oil, liquid paraffin, and olive oil.

An aqueous suspension can be prepared, for example, by admixing at leastone compound of Formula (I) and/or at least one pharmaceuticallyacceptable salt thereof with at least one excipient suitable for themanufacture of an aqueous suspension. Exemplary excipients suitable forthe manufacture of an aqueous suspension, include, but are not limitedto, for example, suspending agents, such as, for example, sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose,sodium alginate, alginic acid, polyvinyl-pyrrolidone, gum tragacanth,and gum acacia; dispersing or wetting agents, such as, for example, anaturally-occurring phosphatide, e.g., lecithin; condensation productsof alkylene oxide with fatty acids, such as, for example,polyoxyethylene stearate; condensation products of ethylene oxide withlong chain aliphatic alcohols, such as, for exampleheptadecaethylene-oxycetanol; condensation products of ethylene oxidewith partial esters derived from fatty acids and hexitol, such as, forexample, polyoxyethylene sorbitol monooleate; and condensation productsof ethylene oxide with partial esters derived from fatty acids andhexitol anhydrides, such as, for example, polyethylene sorbitanmonooleate. An aqueous suspension can also contain at least onepreservative, such as, for example, ethyl and n-propylp-hydroxybenzoate; at least one coloring agent; at least one flavoringagent; and/or at least one sweetening agent, including but not limitedto, for example, sucrose, saccharin, and aspartame.

Oily suspensions can, for example, be prepared by suspending at leastone compound of Formula (I) and/or at least one pharmaceuticallyacceptable salt thereof in either a vegetable oil, such as, for example,arachis oil; olive oil; sesame oil; and coconut oil; or in mineral oil,such as, for example, liquid paraffin. An oily suspension can alsocontain at least one thickening agent, such as, for example, beeswax;hard paraffin; and cetyl alcohol. In order to provide a palatable oilysuspension, at least one of the sweetening agents already describedhereinabove, and/or at least one flavoring agent can be added to theoily suspension. An oily suspension can further contain at least onepreservative, including, but not limited to, for example, ananti-oxidant, such as, for example, butylated hydroxyanisol, andalpha-tocopherol.

Dispersible powders and granules can, for example, be prepared byadmixing at least one compound of Formula (I) and/or at least onepharmaceutically acceptable salt thereof with at least one dispersingand/or wetting agent; at least one suspending agent; and/or at least onepreservative. Suitable dispersing agents, wetting agents, and suspendingagents are as already described above. Exemplary preservatives include,but are not limited to, for example, anti-oxidants, e.g., ascorbic acid.In addition, dispersible powders and granules can also contain at leastone excipient, including, but not limited to, for example, sweeteningagents; flavoring agents; and coloring agents.

An emulsion of at least one compound of Formula (I) and/or at least onepharmaceutically acceptable salt thereof can, for example, be preparedas an oil-in-water emulsion. The oily phase of the emulsions comprisingcompounds of Formula (I) may be constituted from known ingredients in aknown manner. The oil phase can be provided by, but is not limited to,for example, a vegetable oil, such as, for example, olive oil andarachis oil; a mineral oil, such as, for example, liquid paraffin; andmixtures thereof. While the phase may comprise merely an emulsifier, itmay comprise a mixture of at least one emulsifier with a fat or an oilor with both a fat and an oil. Suitable emulsifying agents include, butare not limited to, for example, naturally-occurring phosphatides, e.g.,soy bean lecithin; esters or partial esters derived from fatty acids andhexitol anhydrides, such as, for example, sorbitan monooleate; andcondensation products of partial esters with ethylene oxide, such as,for example, polyoxyethylene sorbitan monooleate. Preferably, ahydrophilic emulsifier is included together with a lipophilic emulsifierwhich acts as a stabilizer. It is also preferred to include both an oiland a fat. Together, the emulsifier(s) with or without stabilizer(s)make-up the so-called emulsifying wax, and the wax together with the oiland fat make up the so-called emulsifying ointment base which forms theoily dispersed phase of the cream formulations. An emulsion can alsocontain a sweetening agent, a flavoring agent, a preservative, and/or anantioxidant. Emulsifiers and emulsion stabilizers suitable for use inthe formulation of the present invention include Tween 60, Span 80,cetostearyl alcohol, myristyl alcohol, glyceryl monostearate, sodiumlauryl sulfate, glyceryl distearate alone or with a wax, or othermaterials well known in the art.

The compounds of Formula (I) and/or at least one pharmaceuticallyacceptable salt thereof can, for example, also be deliveredintravenously, subcutaneously, and/or intramuscularly via anypharmaceutically acceptable and suitable injectable form. Exemplaryinjectable forms include, but are not limited to, for example, sterileaqueous solutions comprising acceptable vehicles and solvents, such as,for example, water, Ringer's solution, and isotonic sodium chloridesolution; sterile oil-in-water microemulsions; and aqueous or oleaginoussuspensions.

Formulations for parenteral administration may be in the form of aqueousor non-aqueous isotonic sterile injection solutions or suspensions.These solutions and suspensions may be prepared from sterile powders orgranules using one or more of the carriers or diluents mentioned for usein the formulations for oral administration or by using other suitabledispersing or wetting agents and suspending agents. The compounds may bedissolved in water, polyethylene glycol, propylene glycol, ethanol, cornoil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodiumchloride, tragacanth gum, and/or various buffers. Other adjuvants andmodes of administration are well and widely known in the pharmaceuticalart. The active ingredient may also be administered by injection as acomposition with suitable carriers including saline, dextrose, or water,or with cyclodextrin (i.e., CAPTISOL®), cosolvent solubilization (i.e.,propylene glycol) or micellar solubilization (i.e., Tween 80).

The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution, and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employed,including synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

A sterile injectable oil-in-water microemulsion can, for example, beprepared by 1) dissolving at least one compound of Formula (I) in anoily phase, such as, for example, a mixture of soybean oil and lecithin;2) combining the Formula (I) containing oil phase with a water andglycerol mixture; and 3) processing the combination to form amicroemulsion.

A sterile aqueous or oleaginous suspension can be prepared in accordancewith methods already known in the art. For example, a sterile aqueoussolution or suspension can be prepared with a non-toxicparenterally-acceptable diluent or solvent, such as, for example,1,3-butane diol; and a sterile oleaginous suspension can be preparedwith a sterile non-toxic acceptable solvent or suspending medium, suchas, for example, sterile fixed oils, e.g., synthetic mono- ordiglycerides; and fatty acids, such as, for example, oleic acid.

Pharmaceutically acceptable carriers, adjuvants, and vehicles that maybe used in the pharmaceutical compositions of this invention include,but are not limited to, ion exchangers, alumina, aluminum stearate,lecithin, self-emulsifying drug delivery systems (SEDDS) such asd-alpha-tocopherol polyethyleneglycol 1000 succinate, surfactants usedin pharmaceutical dosage forms such as Tweens, polyethoxylated castoroil such as CREMOPHOR® surfactant (BASF), or other similar polymericdelivery matrices, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. Cyclodextrins such as alpha-, beta-, and gamma-cyclodextrin,or chemically modified derivatives such as hydroxyalkylcyclodextrins,including 2- and 3-hydroxypropyl-cyclodextrins, or other solubilizedderivatives may also be advantageously used to enhance delivery ofcompounds of the formulae described herein.

The pharmaceutically active compounds of this invention can be processedin accordance with conventional methods of pharmacy to produce medicinalagents for administration to patients, including humans and othermammals. The pharmaceutical compositions may be subjected toconventional pharmaceutical operations such as sterilization and/or maycontain conventional adjuvants, such as preservatives, stabilizers,wetting agents, emulsifiers, buffers etc. Tablets and pills canadditionally be prepared with enteric coatings. Such compositions mayalso comprise adjuvants, such as wetting, sweetening, flavoring, andperfuming agents.

The amounts of compounds that are administered and the dosage regimenfor treating a disease condition with the compounds and/or compositionsof this invention depends on a variety of factors, including the age,weight, sex, the medical condition of the subject, the type of disease,the severity of the disease, the route and frequency of administration,and the particular compound employed. Thus, the dosage regimen may varywidely, but can be determined routinely using standard methods. A dailydose of about 0.001 to 100 mg/kg body weight, preferably between about0.0025 and about 50 mg/kg body weight and most preferably between about0.005 to 10 mg/kg body weight, may be appropriate. The daily dose can beadministered in one to four doses per day. Other dosing schedulesinclude one dose per week and one dose per two day cycle.

For therapeutic purposes, the active compounds of this invention areordinarily combined with one or more adjuvants appropriate to theindicated route of administration. If administered orally, the compoundsmay be admixed with lactose, sucrose, starch powder, cellulose esters ofalkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesiumstearate, magnesium oxide, sodium and calcium salts of phosphoric andsulfuric acids, gelatin, acacia gum, sodium alginate,polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted orencapsulated for convenient administration. Such capsules or tablets maycontain a controlled-release formulation as may be provided in adispersion of active compound in hydroxypropylmethyl cellulose.

Pharmaceutical compositions of this invention comprise at least onecompound of Formula (I) and/or at least one pharmaceutically acceptablesalt thereof, and optionally an additional agent selected from anypharmaceutically acceptable carrier, adjuvant, and vehicle. Alternatecompositions of this invention comprise a compound of the Formula (I)described herein, or a prodrug thereof, and a pharmaceuticallyacceptable carrier, adjuvant, or vehicle.

Utility

The human immune system has evolved to defend the body frommicro-organisms, viruses, and parasites that can cause infection,disease or death. Complex regulatory mechanisms ensure that the variouscellular components of the immune system target the foreign substancesor organisms, while not causing permanent or significant damage to theindividual. While the initiating events are not well understood at thistime, in autoimmune disease states the immune system directs itsinflammatory response to target organs in the afflicted individual.Different autoimmune diseases are typically characterized by thepredominate or initial target organ or tissues affected; such as thejoint in the case of rheumatoid arthritis, the thyroid gland in the caseof Hashimoto's thyroiditis, the central nervous system in the case ofmultiple sclerosis, the pancreas in the case of type I diabetes, and thebowel in the case of inflammatory bowel disease. Thus it has beenobserved that therapeutic agents which act on the immune system orcertain cell types of the immune system (such as B-lymphocytes, and Tlymphocytes, T cells) may have utility in more than one autoimmunedisease.

It is well recognized in the art, including the literature referencescited herein, that S1P receptors are good targets for a wide variety oftherapeutic applications, including autoimmune diseases. S1P receptorsmake good drug targets, because individual receptors are both tissue-and response-specific. Tissue specificity of the S1P receptors isimportant, because development of an agonist or antagonist selective forone receptor localizes the cellular response to tissues containing thatreceptor, limiting unwanted side effects. Response specificity of theS1P receptors is also important because it allows for development ofagonists or antagonists that initiate or suppress certain cellularresponses without affecting other processes. Therefore, compounds thatact on some S1P receptor family members while having diminished or noactivity at other family members are desirable and are expected toprovide a therapeutic effect with an improved side effect profile (i.e.,reduction or elimination of unwanted side effects).

As used herein, the term “agonist” in reference to S1P₁ refers to anagent which exerts pharmacological effects such as decreased motility ofT cells, decreased trafficking of T cells, or decreased egress of Tcells from lymphoid tissues. (Rosen et al., Trends in Immunology, 28:102(2007)).

By virtue of their S1P₁ activity as agonists, the compounds of thepresent invention are immunoregulatory agents useful for treating orpreventing autoimmune or chronic inflammatory diseases. The compounds ofthe present invention are useful to suppress the immune system ininstances where immunosuppression is in order, such as in bone marrow,organ or transplant rejection, autoimmune and chronic inflammatorydiseases, including systemic lupus erythematosis, rheumatoid arthritis,type I diabetes mellitus, inflammatory bowel disease, biliary cirrhosis,uveitis, multiple sclerosis, Crohn's disease, ulcerative colitis,bullous pemphigoid, sarcoidosis, psoriasis, autoimmune myositis,Wegener's granulomatosis, ichthyosis, Graves' ophthalmopathy, andasthma.

More particularly, the compounds of the present invention are useful totreat or prevent a disease or disorder selected from the groupconsisting of: transplantation of organs or tissue, graft-versus-hostdiseases brought about by transplantation, autoimmune syndromesincluding rheumatoid arthritis, juvenile idiopathic arthritis, systemiclupus erythematosus, cutaneous lupus erythematosus (discoid lupuserythematosus, subacute lupus erythematosus) and lupus nephritis,Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type Idiabetes, uveitis, posterior uveitis, allergic encephalomyelitis,glomerulonephritis, post-infectious autoimmune diseases includingrheumatic fever and post-infectious glomerulonephritis, inflammatory andhyperproliferative skin diseases, psoriasis, psoriatic arthritis, atopicdermatitis, contact dermatitis, eczematous dermatitis, seborrhoeicdermatitis, lichen planus, pemphigus, bullous pemphigoid, epidermolysisbullosa, urticaria, angioedemas, vasculitis including ANCA-associatedvasculitis, giant cell arteritis, Takayasu's arteritis, microscopicpoliangiitis, central nervous system vasculitis, Churg-Strauss Syndrome,and rheumatoid vasculitis, erythema, cutaneous eosinophilia, acne,alopecia areata, keratoconjunctivitis, vernal conjunctivitis, uveitisassociated with Behcet's disease, keratitis, herpetic keratitis, conicalcornea, dystrophia epithelialis corneae, corneal leukoma, ocularpemphigus, Mooren's ulcer, scleritis, Graves' ophthalmopathy,Vogt-Koyanagi-Harada syndrome, sarcoidosis, pollen allergies, reversibleobstructive airway disease, bronchial asthma, allergic asthma, intrinsicasthma, extrinsic asthma, dust asthma, chronic or inveterate asthma,late asthma and airway hyper-responsiveness, bronchitis, gastric ulcers,vascular damage caused by ischemic diseases and thrombosis, ischemicbowel diseases, inflammatory bowel diseases, necrotizing enterocolitis,intestinal lesions associated with thermal burns, coeliac diseases,proctitis, eosinophilic gastroenteritis, mastocytosis, Crohn's disease,ulcerative colitis, migraine, rhinitis, eczema, interstitial nephritis,Goodpasture's syndrome, hemolytic-uremic syndrome, diabetic nephropathy,multiple myositis, Guillain-Barre syndrome, Meniere's disease,polyneuritis, multiple neuritis, mononeuritis, radiculopathy,hyperthyroidism, Basedow's disease, pure red cell aplasia, aplasticanemia, hypoplastic anemia, idiopathic thrombocytopenic purpura,autoimmune hemolytic anemia, agranulocytosis, pernicious anemia,megaloblastic anemia, anerythroplasia, osteoporosis, sarcoidosis,fibroid lung, idiopathic interstitial pneumonia, dermatomyositis,leukoderma vulgaris, ichthyosis vulgaris, photoallergic sensitivity,cutaneous T cell lymphoma, arteriosclerosis, atherosclerosis, aortitissyndrome, polyarteritis nodosa, myocardosis, scleroderma, Wegener'sgranuloma, Sjögren's syndrome, adiposis, eosinophilic fascitis, lesionsof gingiva, periodontium, alveolar bone, substantia ossea dentis,glomerulonephritis, male pattern alopecia or alopecia senilis bypreventing epilation or providing hair germination and/or promoting hairgeneration and hair growth, muscular dystrophy, pyoderma and Sezary'ssyndrome, Addison's disease, ischemia-reperfusion injury of organs whichoccurs upon preservation, transplantation or ischemic disease,endotoxin-shock, pseudomembranous colitis, colitis caused by drug orradiation, ischemic acute renal insufficiency, chronic renalinsufficiency, toxinosis caused by lung-oxygen or drugs, lung cancer,pulmonary emphysema, cataracta, siderosis, retinitis pigmentosa, senilemacular degeneration, vitreal scarring, corneal alkali burn, dermatitiserythema multiforme, linear IgA ballous dermatitis and cementdermatitis, gingivitis, periodontitis, sepsis, pancreatitis, diseasescaused by environmental pollution, aging, carcinogenesis, metastasis ofcarcinoma and hypobaropathy, disease caused by histamine orleukotriene-C₄ release, Behcet's disease, autoimmune hepatitis, primarybiliary cirrhosis, sclerosing cholangitis, partial liver resection,acute liver necrosis, necrosis caused by toxin, viral hepatitis, shock,or anoxia, B-virus hepatitis, non-A/non-B hepatitis, cirrhosis,alcoholic cirrhosis, hepatic failure, fulminant hepatic failure,late-onset hepatic failure, “acute-on-chronic” liver failure,augmentation of chemotherapeutic effect, cytomegalovirus infection, HCMVinfection, AIDS, cancer, senile dementia, trauma, neuropathic pain,chronic bacterial infection, thrombocytopenia, IgA nephropathy,mesangioproliferative glomerulonephritis, IgG4-related disease,ankylosing spondylitis, and relapsing polychondritis. Juvenileidiopathic arthritis includes oligoarthritis-onset juvenile idiopathicarthritis, polyarthritis-onset juvenile idiopathic arthritis,systemic-onset juvenile idiopathic arthritis, juvenile psoriaticarthritis, and enthesitis-related juvenile idiopathic arthritis.

One embodiment provides a method for treating autoimmune and/orinflammatory diseases, comprising administering to a mammal in needthereof at least one compound of Formula (I) or a pharmaceuticallyacceptable salt thereof. Another embodiment provides the compounds ofFormula (I) or pharmaceutically acceptable salts thereof, for use intherapy for the treatment of autoimmune and/or inflammatory diseases. Inanother embodiment, provided is the use of the compounds of Formula (I)or pharmaceutically acceptable salts thereof, for the manufacture of amedicament for the treatment or prophylaxis of autoimmune and/orinflammatory disease. A therapeutically effective amount may be employedin these embodiments. Preferably, in these embodiments, the autoimmuneand inflammatory diseases are selected from multiple sclerosis,rheumatoid arthritis, inflammatory bowel disease (including Crohn'sdisease and ulcerative colitis), psoriasis, and as an agent to preventthe rejection of transplanted organs. The method of the presentembodiment includes administration of a therapeutically effect amount ofa compound of Formula (I) or a pharmaceutically effective salt thereof.

In another embodiment, a method for treating vascular disease isprovided comprising administering to a mammal in need thereof at leastone compound of Formula (I) or a pharmaceutically acceptable saltthereof. Another embodiment provides the compounds of Formula (I) orpharmaceutically acceptable salts thereof, for use in therapy for thetreatment of vascular disease. In another embodiment, provided is theuse of the compounds of Formula (I) or pharmaceutically acceptable saltsthereof, for the manufacture of a medicament for treatment of vasculardisease. A therapeutically effective amount may be employed in theseembodiments. Preferably, in these embodiments, the vascular disease isselected from atherosclerosis and ischemia reperfusion injury.

In another embodiment, a method for treating inflammatory bowel diseaseis provided comprising administering to a mammal in need thereof atleast one compound of Formula (I) or a pharmaceutically acceptable saltthereof. Another embodiment provides the compounds of Formula (I) orpharmaceutically acceptable salts thereof, for use in therapy for thetreatment of inflammatory bowel disease. In another embodiment, providedis the use of the compounds of Formula (I) or pharmaceuticallyacceptable salts thereof, for the manufacture of a medicament fortreatment of inflammatory bowel disease. A therapeutically effectiveamount may be employed in these embodiments. Preferably, in theseembodiments, the inflammatory bowel disease is selected from Crohn'sdisease, ulcerative colitis, collagenous colitis, lymphocytic colitis,ischaemic colitis, diversion colitis, Behcet's disease, andindeterminate colitis.

In another embodiment, a method for treating lupus is providedcomprising administering to a mammal in need thereof at least onecompound of Formula (I) or a pharmaceutically acceptable salt thereof.Another embodiment provides the compounds of Formula (I) orpharmaceutically acceptable salts thereof, for use in therapy for thetreatment of lupus. In another embodiment, provided is the use of thecompounds of Formula (I) or pharmaceutically acceptable salts thereof,for the manufacture of a medicament for treatment of lupus. Atherapeutically effective amount may be employed in these embodiments.Lupus includes systemic lupus erythematosus, cutaneous lupuserythematosus, discoid lupus erythematosus, subacute lupus erythematosusand lupus nephritis.

In another embodiment, a method for treating multiple sclerosis isprovided comprising administering to a mammal in need thereof at leastone compound of Formula (I) or a pharmaceutically acceptable saltthereof. Another embodiment provides the compounds of Formula (I) orpharmaceutically acceptable salts thereof, for use in therapy for thetreatment of multiple sclerosis. In another embodiment, provided is theuse of the compounds of Formula (I) or pharmaceutically acceptable saltsthereof, for the manufacture of a medicament for treatment of multiplesclerosis. A therapeutically effective amount may be employed in theseembodiments. Preferably, in these embodiments, multiple sclerosisincludes relapsing remitting multiple sclerosis, primary progressivemultiple sclerosis, secondary progressive multiple sclerosis, andprogressive relapsing multiple sclerosis.

The methods of treating S1P1-associated conditions may compriseadministering compounds of Formula (I) alone or in combination with eachother and/or other suitable therapeutic agents useful in treating suchconditions. Accordingly, “therapeutically effective amount” is alsointended to include an amount of the combination of compounds claimedthat is effective to act as an agonist at the S1P1 receptor. Thecombination of compounds is preferably a synergistic combination.Synergy, as described, for example, by Chou et al., Adv. Enzyme Regul.,22:27-55 (1984), occurs when the effect of the compounds whenadministered in combination is greater than the additive effect of thecompounds when administered alone as a single agent. In general, asynergistic effect is most clearly demonstrated at sub-optimalconcentrations of the compounds. Synergy can be in terms of lowercytotoxicity, increased efficacy, or some other beneficial effect of thecombination compared with the individual components.

Exemplary of such other therapeutic agents include corticosteroids orglucocorticoids such as dexamethasone, methylprednisolone, prednisolone,and prednisone; PDE4 inhibitors such as rolipram, cilomilast,roflumilast, and oglemilast; cytokine-suppressive anti-inflammatorydrugs (CSAIDs) and inhibitors of p38 kinase, 4-substitutedimidazo[1,2-A]quinoxalines as disclosed in U.S. Pat. No. 4,200,750;antibodies or fusion proteins directed to cell surface molecules such asCD2, CD3, CD4, CD8, CD20 such as RITUXAN®, CD25, CD30, CD40, CD69, CD80(B7.1), CD86 (B7.2), CD90, CTLA, for example abatacept (ORENCIA®),belatacept, or their ligands including CD154 (GP39, or CD40L);antibodies to, fusion proteins, or soluble receptors of human cytokinesor growth factors, for example, TNF such as, infliximab (REMICADE®),etanercept (Embrel), adalimumab (HUMIRA®), LT, Il-1 such as anakinra(KINERET®) (an IL-1 receptor antagonist), IL-2, IL-4, IL-5, Il-6, suchas CNTO 328 (a chimeric anti-IL-6 antibody), Il-7, Il-8, Il-12, Il-15,Il-16, Il-17, Il-21, Il-23 such as Ustekinumab (a human anti-IL-12/23monoclonal antibody), and interferons such as interferon beta 1a(AVONEX®, REBIF®), interferon beta 1b (BETASERON®); integrin receptorantagonists such as TYSABRI®; polymeric agents such as glatirameracetate (COPAXONE®); sulfasalazine, mesalamine, hydroxychloroquine,non-steroidal antiinflammatory drugs (NSAIDs) such as salicylatesincluding aspirin, salsalate, and magnesium salicylate, andnon-salicylates such as, ibuprofen, naproxen, meloxicam, celecoxib androfecoxib; antiviral agents such as abacavir; antiproliferative agentssuch as methotrexate, mercaptopurine, leflunomide, cyclosporine,mycophenololate, FK506 (tacrolimus, PROGRAF®); cytotoxic drugs such asazathioprine and cyclophosphamide; nuclear translocation inhibitors,such as deoxyspergualin (DSG); gold containing products such asauronofin; penicllamine, and rapamycin (sirolimus or RAPAMUNE®) orderivatives thereof.

The above other therapeutic agents, when employed in combination withthe compounds of the present invention, may be used, for example, inthose amounts indicated in the Physicians' Desk Reference (PDR) or asotherwise determined by one of ordinary skill in the art. In the methodsof the present invention, such other therapeutic agent(s) may beadministered prior to, simultaneously with, or following theadministration of the inventive compounds.

EXAMPLES

The invention is further defined in the following Examples. It should beunderstood that the Examples are given by way of illustration only. Fromthe above discussion and the Examples, one skilled in the art canascertain the essential characteristics of the invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications to adapt the invention to various uses and conditions.As a result, the invention is not limited by the illustrative examplesset forth hereinbelow, but rather is defined by the claims appendedhereto.

Abbreviations

-   Ac acetyl-   anhyd. anhydrous-   aq. aqueous-   Bn benzyl-   Bu butyl-   Boc tert-butoxycarbonyl-   CV Column Volumes-   DCM dichloromethane-   DEA diethylamine-   DMA N,N-dimethylacetamide-   DMF dimethylformamide-   DMPU 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone-   DMSO dimethylsulfoxide-   EtOAc ethyl acetate-   Et ethyl-   Et₃N triethyl amine-   EtOH ethanol-   H or H₂ hydrogen-   h, hr or hrs hour(s)-   hex hexane-   i iso-   HOAc acetic acid-   HPLC high pressure liquid chromatography-   LC liquid chromatography-   LDA lithium diisopropylamide-   LiHMDS lithium bis(trimethylsilyl)amide-   M molar-   mM millimolar-   Me methyl-   MeCN acetonitrile-   MeOH methanol-   MHz megahertz-   min. minute(s)-   mins minute(s)-   M⁺¹ (M+H)⁺-   MS mass spectrometry-   n or N normal-   nM nanomolar-   NMP N-methylpyrrolidine-   Pd/C palladium on carbon-   Pd₂(dba)₃ tris-(dibenzylideneacetone)dipalladium-   Ph phenyl-   Pr propyl-   PSI pounds per square inch-   R-BINAP (R)-(+)-2,2′-bis(diphenylphosphino)-1,1′-binaphthyl-   Ret Time or RT retention time-   sat. saturated-   S-BINAP (S)-(−)-2,2′-bis(diphenylphosphino)-1,1′-binaphthyl-   SFC supercritical fluid chromatography-   TFA trifluoroacetic acid-   THF tetrahydrofuran    HPLC Conditions:

Condition C: Column: YMC COMBISCREEN® S5 50×4.6 mm (Linear gradient of 0to 100% Solvent B over 4 min, then 1-4 min hold at 100% B; SolventA=water 90%/MeOH 10%/H₃PO₄, 0.2%; Solvent B=MeOH 90%/water 10%/H₃PO₄0.2%. Flow rate: 4 mL/min; Products detected at 220 nm.

Condition G: Column: Waters Acquity BEH C18 2.1×50 mm 1.7 μm; Lineargradient of 0-100% Solvent B over 3 min, then 0.75 min hold at 100% B;Flow rate: 1.11 mL/min; Solvent A: 5:95 acetonitrile:water with 10 mMammonium acetate; Solvent B: 95:5 acetonitrile:water with 10 mM ammoniumacetate; Temperature=50° C.; Products detected at 220 nm wavelength.

Condition H: Column: SunFire C18, (150×3.0 mm), 3.5 μm; Linear gradientof 10 to 100% Solvent B over 25 min, then 5 min hold at 100% B; Flowrate: 1 mL/min; Buffer: 0.5% TFA, in water with pH adjusted to 2.5 usingdilute ammonia; Solvent A: Buffer: acetonitrile (95:5); Solvent B:Buffer: acetonitrile (5:95); Products detected at 220 nm.

Condition I: Column: Waters Acquity SD BEH C18, 2.1×50 mm, 1.7-μmparticles; Mobile Phase A: 100% H₂O with 0.05% TFA; Mobile Phase B: 100%acetonitrile with 0.05% TFA; Temperature: 50° C.; gradient of 98:2 to2:98 (A %:B %) over 1 min and hold 2:98 for 0.5 min.; Flow: 0.800 mL/minProducts detected at 220 nm.

Condition J: Column: CHROMOLITH® SpeedROD (4.6×50 mm); Linear gradientof 0 to 100% Solvent B over 4 min, with 1 min hold at 100% B; Solvent A:10% MeOH, 90% H₂O, 0.1% TFA; Solvent B: 90% MeOH, 10% H₂O, 0.1% TFA;Flow rate: 4 mL/min; Products detected at 220 nm.

Intermediate 1 (1R,3S)-Methyl1-amino-3-(4-bromophenyl)cyclopentanecarboxylate

Intermediate 1A: (S)-3-(4-Bromophenyl)cyclopentanone

A solution of 4-bromophenylboronic acid (20 g, 100 mmol) in 1,4-dioxane(120 mL) in a 500 ml flask was purged with nitrogen for 5 mins. S-BINAP(0.992 g, 1.593 mmol) and bis(norbornadiene)rhodium (I)tetrafluoroborate (0.559 g, 1.494 mmol) were added sequentially to thesolution under a positive pressure of nitrogen. After 2 hours ofagitation at room temperature, water (20 mL) was added followed bycyclopent-2-enone (8.06 mL, 100 mmol) and Et₃N (13.88 mL, 100 mmol). Themixture was allowed to stir at room temperature for 16 hours. Theresulting dark solids were removed by filtration and the filtrate waspoured into 250 ml of ethyl acetate. The solution was washed with watertwice and the organic layer was concentrated. The residue was purifiedby flash column chromatography (split into two batches, each run on a330 g silica column 0%-25% ethyl acetate in hexane) to afford 12.1 gramsof (5)-3-(4-bromophenyl)cyclopentanone. HPLC purity was >98% and ChiralHPLC analysis indicated approximately 90% ee. The material was furtherpurified under the Chiral SFC conditions described below. ExperimentalDetails: Instrument: Berger SFC MGIII; Preparative Conditions: Column:CHIRALPAK® AD-H 25×5 cm, 5 μm; Column Temperature: 40° C.; Flow rate:200 mL/min; Mobile Phase: CO₂/MeOH=80/20; Detector Wavelength: 225 nm;Injection Vol.: 1.0 mL; Sample Preparation: 12.1 g in; 210 mL MeOH(Conc. 60 mg/ml); Analytical Conditions: Column: CHIRALPAK® AD 25×0.46cm, 10 μm; Column Temperature: 40° C.; Flow rate: 2.0 min; Mobile Phase:CO₂/MeOH=70/30; Detector Wavelength: 220 nm; Injection Vol.: 5 μL.

The desired enantiomer (major isomer) was isolated and named as “PK2”based on the elution order. The enantiomeric purity of the isolatedisomer was determined to be greater than 99.6% on SFC/UV area % at 220nm. After evaporation, 10.5 grams of the desired enantiomer wererecovered. HPLC retention time=LC/MS M⁺¹=239/241. ¹H NMR (400 MHz,CD₃OD) δ ppm 7.43-7.51 (2H, m), 7.10-7.19 (2H, m), 3.32-3.46 (1H, m),2.67 (1H, dd, J=18.27, 7.48 Hz), 2.39-2.54 (2H, m), 2.23-2.39 (2H, m),1.97 (1H, ddd, J=12.98, 11.00, 9.02 Hz).

Intermediate 1B:(7S)-7-(4-Bromophenyl)-1,3-diazaspiro[4.4]nonane-2,4-dione

A total of 9.8 g (S)-3-(4-bromophenyl)cyclopentanone was used, dividedinto 2 batches each containing 4.9 g. The two batches were processedunder identical conditions as described below.

To a mixture of (S)-3-(4-bromophenyl)cyclopentanone (I-1A, 4.9 g, 20.49mmol) and potassium cyanide (1.935 g, 29.7 mmol) in EtOH (40 mL) andwater (20 mL) in a glass pressure vessel was added ammonium carbonate(4.92 g, 51.2 mmol). The reaction vessel was sealed and placed in an oilbath heated at 80° C. for 24 hours, resulting in the formation of awhite solid. After cooling the reaction vessel in an ice bath, thevessel was opened and 30 ml of water was added resulting in theformation of additional solids. The solids were collected by filtration,washed twice with 5 ml water, and then dried under high vacuum. The twobatches were combined to provide 13.9 g of(7S)-7-(4-bromophenyl)-1,3-diazaspiro[4.4]nonane-2,4-dione) which wereused for subsequent reactions without further purification. HPLCretention time=0.82 min (Condition G) LC/MS M^(+Na)=331, 2M^(+H)=619.

Intermediate 1C: (3S)-1-Amino-3-(4-bromophenyl)cyclopentanecarboxylicacid

To (7S)-7-(4-bromophenyl)-1,3-diazaspiro[4.4]nonane-2,4-dione (I-1B,13.9 g, 45.0 mmol) in 1,4-dioxane (40 mL) in a round bottom flask wasadded aqueous NaOH (2N, 100 mL, 200 mmol). The mixture was heated to 95°C. and stirred for 24 hours. Additional NaOH (25 mL, 50 mmol) was addedand heating continued for another two days. The solution was cooled withan ice-bath, neutralized with 5N HCl to approximately pH 7 resulting inthe formation of a white precipitate. The solids were collected byfiltration and dried under high vacuum for 2 days to provide 14 g of(3S)-1-amino-3-(4-bromophenyl)cyclopentanecarboxylic acid as a whitesolid which was used as such for the subsequent step without furtherpurification. HPLC retention time=0.64 min (Condition G) LC/MSM⁺¹=284/286.

Intermediate 1D: (3S)-Methyl1-amino-3-(4-bromophenyl)cyclopentanecarboxylate

To a heterogeneous mixture of(3S)-1-amino-3-(4-bromophenyl)cyclopentanecarboxylic acid (I-1C, 14 g,49.3 mmol) in MeOH (250 mL) was added thionyl chloride (36.0 mL, 493mmol) dropwise over a period of 20 minutes at room temperature via anadditional funnel (exothermic). The reaction mixture was placed in anoil bath and heated to 70° C. for 4 hours. The solvent was removed undervacuum, with the residue being dissolved in ethyl acetate (200 mL) andwashed twice with 1N NaOH. The organic layer was then dried over Na₂SO₄and concentrated to give 10.8 g of (3S)-methyl1-amino-3-(4-bromophenyl)cyclopentanecarboxylate. HPLC retentiontime=0.68 min (Condition G); LC/MS M⁺¹=298/300.

Intermediate 1: (1R,3S)-Methyl1-amino-3-(4-bromophenyl)cyclopentanecarboxylate

The mixture of diastereomers (I-1D, 9.5 g) was separated by Chiral SFC.The absolute stereochemical assignment of Intermediate 1 and itsdiastereomer was previously described (Wallace, G. A. et al., J. Org.Chem., 74:4886-4889 (2009)). Experimental Details: Instrument:Preparative: Thar SFC350; Analytical: Berger analytical SFC; PreparativeConditions: Column: Lux-Cellulose-4 25×3 cm, 5 μm; Column Temperature:35° C.; Flow rate: 200 ml/min; Mobile Phase: CO₂/(MeOH with 0.1%DEA)=87/13; Detector Wavelength: 220 nm; Injection Vol.: 0.6 ml; SamplePreparation: 9.5 g in 400 ml MeOH (Conc. 23.7 mg/ml). AnalyticalConditions: Column: Lux-Cellulose-4 25×0.46 cm, 5 μm; Column Temp. 35°C.; Flow rate: 3 ml/min; Mobile Phase: CO₂/(MeOH with 0.1% DEA)=85/15;Detector Wavelength: 220 nm; Injection Vol.: 5 μL.

Intermediate 1: Peak 2: 4.06 g; ret. time=6.64 min on the analyticalchiral SFC conditions above. Optical purity: 98.2%; LC/MS M⁺¹=298/300;Peak 1: 3.96 g; ret. time=5.47 min on the analytical chiral SFCconditions above. Optical purity: 99.4%.

Alternative Preparation: HCl Salt of Intermediate 1

A solution of (3S)-1-amino-3-(4-bromophenyl)cyclopentanecarboxylic acid(10.2 g, 35.9 mmol) in MeOH (100 mL) was cooled in an ice bath, followedby addition of SOCl₂ (15.72 mL, 215 mmol) dropwise. After the additionwas complete, the solution was refluxed for 3 hrs at which time thereaction was determined to be complete by HPLC. The solution wasconcentrated to remove methanol to afford a solid. The solid was takenin 50 ml of 3% H₂O in EtOAc and stirred well for 30 mins. The whitesolid formed was collected by filtration. The wet white solid was takenin 50 ml of 4% H₂O in 1,2-dimethoxyethane and heated to 50° C. for 3hrs, and then stirred at room temperature overnight. The resulting whitesolid was collected by filtration and dried to afford (1R,3S)-methyl1-amino-3-(4-bromophenyl)cyclopentanecarboxylate hydrochloride (3.5 g,10.35 mmol). HPLC retention time=6.6 min (Condition H) LC/MSM⁺¹=298/300. ¹H NMR (400 MHz, DMSO-d₆) δ 8.95 (br. s, 3H) 7.50-7.53 (m,2H), 7.35-7.37 (m, 2H), 3.81 (s, 3H) 3.17-3.28 (m, 1H), 2.57 (dd, J=14,7 Hz, 1H), 2.0-2.28 (m, 5H).

Intermediate 2 (1R,3R)-Methyl1-amino-3-(4-bromophenyl)cyclopentanecarboxylate

Intermediate 2A: (R)-3-(4-Bromophenyl)cyclopentanone

A solution of 4-bromophenylboronic acid (20 g, 100 mmol) in 1,4-dioxane(120 mL) was purged with nitrogen for 10 min. (R)-BINAP (0.992 g, 1.593mmol) and bis(norbornadiene)rhodium (I) tetrafluoroborate (0.559 g,1.494 mmol) were added sequentially, and the suspension was sonicatedfor 5 min. The mixture was stirred for 20 min. Water (20 mL) was added,and the reaction mixture became homogeneous. After 10 minutes,cyclopent-2-enone (8.06 mL, 100 mmol) was added, and the reactionmixture was stirred at room temperature overnight. HPLC and LCMSanalysis indicated that the reaction had proceeded, but there was morestarting material than product. The reaction mixture was filteredthrough a pad of CELITE®, and the CELITE® was washed with ethyl acetate(100 mL). The filtrate was diluted with an additional ethyl acetate (150mL), washed with water (2×), washed with brine, and dried over anhydroussodium sulfate. The product mixture was purified by flash silica gelchromatography using a mixture of ethyl acetate and hexane to give(R)-3-(4-bromophenyl)cyclopentanone (6.09 g, 25.5 mmol) as a whitesolid. The product was 98% pure by HPLC with a retention time=2.11min.—(Condition J). LC/MS M⁺¹=241 ¹H NMR (400 MHz, chloroform-d) δ7.57-7.39 (m, 2H), 7.22-7.06 (m, 2H), 3.39 (ddd, J=10.9, 6.8, 4.1 Hz,1H), 2.67 (dd, J=18.2, 7.4 Hz, 1H), 2.57-2.38 (m, 2H), 2.38-2.21 (m,2H), 1.99-1.85 (m, 1H).

Chiral HPLC indicated that the compound was 90-95% enantiomericallypure. The compound (6.03 g) was further purified by Chiral SFC using theconditions listed below. The desired enantiomer was isolated and namedas “PK1” in the elution order. The enantiomeric purity of the isolatedisomer was determined to be greater than 99.9% on SFC/UV area % at 220nm. 5.45 grams of the desired enantiomer was recovered afterconcentration. Experimental Details: Instrument: Berger SFC MGIII; Prep.Conditions; Column: CHIRALPAK® AD-H 25×3 cm, 5 μm; Column Temperature:40° C.; Flow rate: 180 mL/min; Mobile Phase: CO₂/MeOH=87/13; DetectorWavelength: 225 nm; Injection Vol.: 0.5 mL; Sample Preparation: 6.03 gin 100 mL MeOH (Conc. 60 mg/ml). Analytical Conditions: Column:CHIRALPAK® AD 25×0.46 cm, 10 μm; Column Temperature: 40° C.; Flow rate:2.0 min; Mobile Phase: CO₂/MeOH=70/30; Detector Wavelength: 220 nm;Injection Vol.: 5 μL.

Intermediate 2B:(7R)-7-(4-Bromophenyl)-1,3-diazaspiro[4.4]nonane-2,4-dione

To a mixture of (R)-3-(4-bromophenyl)cyclopentanone (I-2A, 5.4 g, 22.58mmol) and potassium cyanide (2.132 g, 32.7 mmol) in EtOH (40 mL) andwater (20 mL) in a glass pressure vessel was added ammonium carbonate(5.42 g, 56.5 mmol). The reaction vessel was sealed and placed in an oilbath heated at 80° C. for 20 hours. A large amount of white, freeflowing solid formed in the pale yellow solution. Analysis by LCMSindicated remaining starting material so the reaction was continued foran additional 24 hours. As conversion was incomplete, the temperature ofthe oil bath was raised to 120° C. The white solid completely dissolvedat the higher temperature. After 3 hours the solution was cooled down toroom temperature. The solution was further cooled in an ice bath, water(30 mL) was added and the resulting white solid was collected byfiltration, washed with water, air dried, then placed under high vacuumto afford the target compound (6.9 g, 22.32 mmol) which was used forsubsequent reaction without additional purification. HPLC retentiontime=0.81 min (Condition G); LC/MS M^(H)=309/311; 2M^(+H)=619.

Intermediate 2C: (3R)-1-Amino-3-(4-bromophenyl)cyclopentanecarboxylicacid

A solution of (7R)-7-(4-bromophenyl)-1,3-diazaspiro[4.4]nonane-2,4-dione(I-2B, 6.80 g, 22 mmol) in dioxane (20 mL) and NaOH (2N aq) (120 mL, 240mmol) was heated in an oil bath set to 95° C. The resulting clear, paleyellow solution was left to stir over the weekend. The solution wascooled in an ice bath and neutralized to approximately pH 7 with 6 N HClresulting in the formation of a precipitate. The solids were collectedand left to air dry overnight. The white solid was slurried in hotethanol (˜100 mL) and re-collected by filtration and the solid wasair-dried then placed under high vacuum. (5.8 g, 20.41 mmol). HPLCretention time=0.64 min (Condition G); LC/MS M⁺¹=284/286. ¹H NMR (500MHz, methanol-d₄) δ 7.52-7.38 (m, 2H), 7.31-7.17 (m, 2H), 3.55-3.40 (m,1H), 2.68 (dd, J=13.3, 6.7 Hz, 1H from single diastereomer), 2.58-2.39(m, 1H), 2.26-2.15 (m, 1H), 2.10-1.98 (m, 1H), 1.98-1.81 (m, 1H), 1.70(dd, J=13.2, 11.8 Hz, 1H from single diastereomer).

Intermediate 2D: (3R)-Methyl1-amino-3-(4-bromophenyl)cyclopentanecarboxylate

In a 500 mL round bottom flask containing a stir bar,(3R)-1-amino-3-(4-bromophenyl)cyclopentanecarboxylic acid (I-2C, 5.4 g,19.00 mmol) was suspended in methanol (100 mL) to afford a white slurry.A dropping funnel was charged with thionyl chloride (13.87 mL, 190 mmol)and the reagent was added dropwise at a rate to keep the mixture fromreaching reflux temperature. After the addition was complete, the paleyellow, milky solution was placed in an oil bath set to 70° C. and anair-cooled reflux condenser was attached. The solution was heated forseveral hours and then allowed to cool to room temperature overnight.The solvent was evaporated under vacuum. The residue was dissolved inethyl acetate, washed with 1N NaOH (aq), washed with water, then driedover MgSO₄ before being filtered and concentrated. The resulting yellowsolid was slurried in warm ethyl acetate, with sonication and thenfiltered. The solid was air-dried and placed under vacuum and thefiltrate was evaporated to afford Solid 1: white solid, 4.28 g LCMSshows >98% AP. The filtrate was evaporated to afford a yellow solid(1.89 g). The solid from the filtrate was slurried in a minimal amountof hot ethyl acetate, with sonication, then cooled (ice bath) andfiltered cold. The solid was air-dried and placed under vacuum to affordSolid 2: 1.44 g white solid. Combined solids (5.7 g).

Intermediate 2: (1R,3R)-Methyl1-amino-3-(4-bromophenyl)cyclopentanecarboxylate

The combined solids of (3R)-methyl1-amino-3-(4-bromophenyl)cyclopentanecarboxylate (I-2D, 4 g) wereseparated using Chiral SFC separation of the diastereomers. The absolutestereochemical assignment of Intermediate 2 and its diastereomer hasbeen previously described (Wallace, G. A. et al., J. Organic Chem.,74:4886-4889 (2009)). Experimental Details: Instrument: Preparative:Thar SFC350; Analytical: Thar analytical MDS. Preparative Conditions:Column: CHIRALPAK® AD-H 25×5 cm, 5 μm; Column Temperature: 35° C.; Flowrate: 300 ml/min; Mobile Phase: CO₂/(MeOH with 0.1% DEA)=82/18; DetectorWavelength: 230 nm; Injection Vol.: 0.4-0.5 ml; Sample Preparation: 4 gin 120 ml MeOH (Conc. 33 mg/ml). Analytical Conditions: Column:CHIRALPAK® AD-H 25×0.46 cm, 5 μm; Column Temperature: 35° C.; Flow rate:3 ml/min; Mobile Phase: CO₂/(MeOH with 0.1% DEA)=80/20; DetectorWavelength: 222 nm; Injection Vol.: 5 μL.

Intermediate 2 (Peak 1): 1.56 g (99.3% optical purity at 222 nm) Ret.Time=7.18 min on analytical chiral SFC. ¹H NMR (500 MHz, methanol-d₄) δ7.45-7.39 (m, 2H), 7.23-7.17 (m, 2H), 3.78 (s, 3H), 3.40-3.48 (m, 1H),2.40 (ddd, J=13.0, 8.9, 3.6 Hz, 1H), 2.28-2.21 (m, 1H), 2.18 (dd,J=13.0, 11.7 Hz, 1H), 2.04 (dd, J=13.0, 7.2 Hz, 1H), 1.88-1.79 (m, 1H),1.79-1.70 (m, 1H).

Peak 2: 1.8 g (97.2% optical purity at 222 nm). Ret. Time=7.71 min onanalytical chiral SFC. ¹H NMR (500 MHz, methanol-d₄) δ 7.45-7.38 (m,2H), 7.26-7.20 (m, 2H), 3.78 (s, 3H), 3.28-3.20 (m, 1H), 2.66-2.57 (m,1H), 2.25 (ddd, J=12.8, 11.0, 7.2 Hz, 1H), 2.10 (dt, J=12.2, 6.8 Hz,1H), 2.03-1.93 (m, 1H), 1.84 (ddd, J=13.0, 7.8, 2.2 Hz, 1H), 1.65 (dd,J=13.3, 11.1 Hz, 1H).

Intermediate 3(5R,7S)-7-(4-Bromophenyl)-3-oxa-1-azaspiro[4.4]nonan-2-one

Intermediate 3A: ((1R,3S)-1-Amino-3-(4-bromophenyl)cyclopentyl)methanol

To a mixture of (1R,3S)-methyl1-amino-3-(4-bromophenyl)cyclopentanecarboxylate, HCl (I-1 HCl, 15 g,44.8 mmol) in MeOH (100 mL) at 0° C. was added sodium borohydride (4 g,106 mmol) portionwise. The reaction mixture was warmed to roomtemperature and sodium borohydride was added portionwise until thereaction was determined to be complete by HPLC analysis. Water was addedto quench the reaction. The reaction mixture was diluted with ethylacetate and washed with saturated NaCl. The aqueous layer was backextracted several times. The combined organic layers were dried withMgSO₄, filtered and concentrated. The product (11 g) was recovered afterconcentration. HPLC retention time=0.65 min (Condition G); LC/MSM⁺¹=272: ¹H NMR (400 MHz, DMSO-d₆) δ 7.51-7.40 (m, 2H), 7.27 (d, J=8.4Hz, 2H), 3.32-3.20 (m, 2H), 3.09-2.92 (m, 1H), 2.11 (dd, J=12.9, 8.7 Hz,1H), 1.98-1.87 (m, 1H), 1.80 (qd, J=11.1, 7.9 Hz, 1H), 1.69-1.58 (m,1H), 1.48 (ddd, J=12.4, 7.9, 2.2 Hz, 1H), 1.32 (dd, J=12.8, 10.1 Hz,1H).

Intermediate 3:(5R,7S)-7-(4-Bromophenyl)-3-oxa-1-azaspiro[4.4]nonan-2-one

To a mixture of ((1R,3S)-1-amino-3-(4-bromophenyl)cyclopentyl)methanol(11 g, 40.7 mmol) and pyridine (I-3A, 3.29 mL, 40.7 mmol) in dioxane(300 mL) was added 1,1′-carbonyldiimidazole (19.81 g, 122 mmol). Thereaction mixture was stirred for 4 hours. The reaction mixture wasdiluted with ethyl acetate and washed with 1M HCl, brine and saturatedNaHCO₃. The mixture was back extracted several times. The organic layerwas dried with MgSO₄, filtered and concentrated to afford 10.5 g ofdesired product as an off-white solid. HPLC retention time=0.87 min(Condition G). LC/MS M⁺¹=297.9; ¹H NMR (400 MHz, chloroform-d) δ 7.45(d, J=8.6 Hz, 2H), 7.12 (d, J=8.4 Hz, 2H), 6.42 (br. s., 1H), 4.41-4.21(m, 2H), 3.17-2.91 (m, 1H), 2.34 (dd, J=13.3, 7.4 Hz, 1H), 2.23-2.11 (m,2H), 2.01-1.90 (m, 2H), 1.88-1.74 (m, 1H).

Intermediate 4(5R,7R)-7-(4-Bromophenyl)-3-oxa-1-azaspiro[4.4]nonan-2-one

Intermediate 4A: ((1R,3R)-1-Amino-3-(4-bromophenyl)cyclopentyl)methanol

(1R,3R)-Methyl 1-amino-3-(4-bromophenyl)cyclopentanecarboxylate (1-2,3.88 g, 13.01 mmol) was dissolved in MeOH (65.1 ml) and sodiumborohydride (1.477 g, 39.0 mmol) was added portionwise. Additionalsodium borohydride was added (0.5 equiv every 1 h) portionwise until thereaction was determined to be complete by HPLC analysis. The reactionwas found to be complete after 2 hours. The reaction mixture wasquenched with water and diluted with ethyl acetate. The aqueous layerwas back extracted three times with EtOAc. The organic layers werecombined, washed with saturated NaCl, dried over MgSO₄, filtered andconcentrated to afford((1R,3R)-1-amino-3-(4-bromophenyl)cyclopentyl)methanol (3.19 g, 11.81mmol). HPLC ret time=0.68 min; LC/MS M⁺¹=272: ¹H NMR (400 MHz, CDCl₃) δ7.42 (d, J=8.4 Hz, 2H), 7.13 (d, J=8.4 Hz, 2H), 3.49 (s, 2H), 3.32-3.41(m, 1H), 2.19-2.25 (m, 1H), 1.98-2.07 (m, 1H), 1.90-1.95 (m, 1H),1.66-1.74 (m, 2H), 1.52-1.60 (m, 1H).

Intermediate 4:(5R,7R)-7-(4-Bromophenyl)-3-oxa-1-azaspiro[4.4]nonan-2-one

((1R,3R)-1-Amino-3-(4-bromophenyl)cyclopentyl)methanol (I-4A, 3.19 g,11.81 mmol) was dissolved in THF (59.0 ml). Pyridine (0.955 ml, 11.81mmol) and 1,1′-carbonyldiimidazole (5.74 g, 35.4 mmol) were addedportionwise. The reaction mixture was stirred for 4 h and was followedby LCMS. After completion, the mixture was diluted with EtOAc and washedwith 1M HCl. The aqueous layer was back extracted twice with EtOAc. Theorganic layers were combined, washed with saturated NaCl, dried overMgSO₄, filtered and concentrated to afford(5R,7R)-7-(4-bromophenyl)-3-oxa-1-azaspiro[4.4]nonan-2-one (2.5 g, 8.44mmol) after flash chromatography (24 g silica gel column; eluent: hexane2 CV followed by a gradient to 100% EtOAc over 15 CV). HPLC rettime=0.91 min; LC/MS M⁺¹=298. ¹H NMR (400 MHz, CDCl₃) δ 7.46 (d, J=8.5Hz, 2H), 7.09 (d, J=8.5 Hz, 2H), 5.72-5.81 (m, 1H), 4.35 (dd, J=13 Hz, 8Hz, 2H), 3.19-3.24 (m, 1H), 2.38-2.44 (m, 1H), 2.15-2.26 (m, 1H),2.11-2.14 (m, 1H), 1.99-2.05 (m, 1H), 1.79-1.85 (m, 1H), 1.65-1.72 (m,1H).

Intermediate 5(5R,7S)-7-(6-Oxo-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one

Intermediate 5A: tert-Butyl2-(4-((5R,7S)-2-oxo-3-oxa-1-azaspiro[4.4]nonan-7-yl)phenyl)acetate

To a mixture of(5R,7S)-7-(4-bromophenyl)-3-oxa-1-azaspiro[4.4]nonan-2-one (I-3, 1 g,3.38 mmol) in dioxane (10 mL) at room temperature was added lithiumbis(trimethylsilyl)amide (3.71 mL, 3.71 mmol). The mixture was stirredfor 30 minutes, then1,2,3,4,5-pentaphenyl-1′-(di-t-butylphosphino)ferrocene (0.121 g, 0.169mmol), Pd₂(dba)₃ (0.155 g, 0.169 mmol) and(2-(tert-butoxy)-2-oxoethyl)zinc(II) chloride (8.10 mL, 4.05 mmol) wereadded. The reaction mixture was heated at 80° C. for 2 hours, thencooled to room temperature, diluted with ethyl acetate and washed with1M HCl. The organic layer was dried with MgSO₄, filtered andconcentrated. The crude material was purified on a silica gel cartridge(40 g) using an EtOAc/hexane gradient (0-100% EtOAc over 20 minutes) toafford 950 mg of tert-butyl2-(4-((5R,7S)-2-oxo-3-oxa-1-azaspiro[4.4]nonan-7-yl)phenyl)acetate. HPLCretention time=0.93 min (Condition G); LC/MS M⁺¹=332.

Intermediate 5B:2-(4-((5R,7S)-2-Oxo-3-oxa-1-azaspiro[4.4]nonan-7-yl)phenyl)acetic acid

To a mixture of tert-butyl2-(4-((5R,7S)-2-oxo-3-oxa-1-azaspiro[4.4]nonan-7-yl)phenyl)acetate(I-5A, 1 g, 3.02 mmol) in DCM (20 mL) was added TFA (10 mL). After 2 h,the solution was concentrated in vacuo and used as such for thesubsequent step without further purification. HPLC retention time=0.65min (Condition G); LC/MS M⁺¹=276.

Intermediate 5:(5R,7S)-7-(6-Oxo-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one

To a mixture of2-(4-((5R,7S)-2-oxo-3-oxa-1-azaspiro[4.4]nonan-7-yl)phenyl)acetic acid(I-5B, 800 mg, 2.91 mmol) in DCM (20 mL) was added oxalyl chloride (1ml, 11.42 mmol) and a few drops of DMF. After one hour, the reactionmixture was concentrated in vacuo. The residue was re-dissolved in DCM(20 mL) in a glass pressure vessel. Granular aluminum chloride (1550 mg,11.62 mmol) was added and the reaction mixture was cooled to −78° C.Ethylene was bubbled through the solution for 5 minutes and then thereaction vessel was sealed. The reaction mixture was allowed to slowlywarm to room temperature and stirred for 4 hours. The mixture was pouredonto ice, diluted with dichloromethane and washed with 1M HCl. Theorganic layer was dried with MgSO₄, filtered and concentrated. The crudematerial was purified on a silica gel cartridge (80 g) using a MeOH/DCMgradient (0-10% MeOH over 13 CV). The product containing fractions werecollected and dried in vacuo to afford 770 mg of(5R,7S)-7-(6-oxo-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one.HPLC retention time=0.74 min (Condition G); LC/MS M⁺¹=286: ¹H NMR (400MHz, chloroform-d) δ 7.20-7.00 (m, 3H), 5.49 (br. s., 1H), 4.45-4.25 (m,2H), 3.59 (s, 2H), 3.08 (t, J=6.8 Hz, 3H), 2.58 (t, J=6.7 Hz, 2H), 2.38(dd, J=13.2, 7.3 Hz, 1H), 2.27-2.11 (m, 2H), 2.05-1.92 (m, 2H),1.92-1.74 (m, 1H).

Intermediate 66-((5R,7S)-2-Oxo-3-oxa-1-azaspiro[4.4]nonan-7-yl)-3,4-dihydronaphthalen-2-yltrifluoromethanesulfonate

To a mixture of(5R,7S)-7-(6-oxo-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one(1-5, 340 mg, 1.192 mmol) and DMPU (0.431 mL, 3.57 mmol) in THF (10 mL)at −78° C. was added LDA (1.456 mL, 2.62 mmol). The reaction mixture wasstirred for 30 minutes then1,1,1-trifluoro-N-phenyl-N-(trifluoromethyl)sulfonyl methanesulfonamide(639 mg, 1.787 mmol) in THF (10 mL) was added. The reaction mixture waswarmed to 0° C. After 1 hour, the reaction was quenched with water. Thereaction mixture was diluted with ethyl acetate and washed withsaturated aqueous NaCl. The organic layer was dried with MgSO₄, filteredand concentrated. The crude material was purified on a silica gelcartridge (40 g) using an EtOAc/hexane gradient (0-100% EtOAc over 20minutes) to afford 400 mg of6-((5R,7S)-2-oxo-3-oxa-1-azaspiro[4.4]nonan-7-yl)-3,4-dihydronaphthalen-2-yltrifluoromethanesulfonate.HPLC retention time=1.01 min (Condition G); LC/MS M⁺¹=418. ¹H NMR (400MHz, chloroform-d) δ 7.17-6.95 (m, 3H), 6.74 (s, 1H), 6.48 (s, 1H),4.48-4.20 (m, 2H), 3.17-2.95 (m, 3H), 2.81-2.60 (m, 2H), 2.33 (dd,J=13.3, 7.2 Hz, 1H), 2.24-2.08 (m, 2H), 2.05-1.74 (m, 3H).

Intermediate 7(5R,7R)-7-(6-Oxo-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one

Intermediate 7A: tert-Butyl2-(4-((5R,7R)-2-oxo-3-oxa-1-azaspiro[4.4]nonan-7-yl)phenyl)acetate

To a solution of(5R,7R)-7-(4-bromophenyl)-3-oxa-1-azaspiro[4.4]nonan-2-one (Int. 4, 2.1g, 7.09 mmol) in THF (25.3 ml) at room temperature was added LiHMDS(7.80 ml, 7.80 mmol). The solution was stirred for 15 min. Next,Pd₂(dba)₃ (0.195 g, 0.213 mmol),1,2,3,4,5-pentaphenyl-1′-(di-t-butylphosphino)ferrocene (0.151 g, 0.213mmol), and (2-(tert-butoxy)-2-oxoethyl)zinc(II) bromide, tetrahydrofuran(7.07 g, 21.27 mmol) were sequentially added. The slurry was stirred at24° C. for 2 h. LCMS analysis showed complete consumption of thestarting material. The reaction mixture was diluted with ethyl acetateand washed with 1M HCl. The organic layer was dried over MgSO₄, filteredand concentrated. The crude material was purified on a silica gelcartridge (40 g) using hexane: acetone 100:0 to 0:100 over 25 CV.tert-Butyl2-(4-((5R,7R)-2-oxo-3-oxa-1-azaspiro[4.4]nonan-7-yl)phenyl)acetate (2.35g, 7.09 mmol) was isolated. HPLC retention time=0.95 min (Condition I):LC/MS M⁺¹=332: ¹H NMR (400 MHz, chloroform-d) δ 7.27-7.21 (m, 2H),7.21-7.15 (m, 2H), 5.11 (br. s., 1H), 4.40-4.26 (m, 2H), 3.53 (s, 2H),3.22-3.01 (m, 1H), 2.36 (dd, J=13.2, 7.3 Hz, 1H), 2.25-2.10 (m, 2H),2.04-1.92 (m, 2H), 1.91-1.76 (m, 1H), 1.47 (s, 9H).

Intermediate 7:(5R,7R)-7-(6-Oxo-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one

The brown liquid tert-butyl2-(4-((5R,7R)-2-oxo-3-oxa-1-azaspiro[4.4]nonan-7-yl)phenyl)acetate(I-7A, 2.35 g, 7.09 mmol) was dissolved in DCM (60 mL) followed by theaddition of trifluoroacetic acid (20 mL, 260 mmol). The reaction mixturewas stirred at room temperature for 1 h at which time the solvent wasremoved under reduced pressure. The resulting material was diluted inDCM (60 mL), purified by acid/base extraction and placed under vacuumfor 1 h. The resulting brown gum2-(4-((5R,7R)-2-oxo-3-oxa-1-azaspiro[4.4]nonan-7-yl)phenyl)acetic acid(1.952 g, 7.09 mmol) was dissolved in DCM (60 mL) followed by theaddition of oxalyl chloride (1.862 mL, 21.27 mmol), and DMF (0.027 mL,0.355 mmol). The resulting solution was stirred until the evolution ofgas ceased (about 30 min) at room temperature. LCMS of an aliquotquenched with MeOH showed complete consumption of the acid (RT=0.65 min,Condition I) and appearance of the presumed methyl ester due to methanolquench (RT=0.77 min, Condition I) as the only product. The solvent wasremoved under reduced pressure and the product was placed under vacuum.The brown gum was transfer to a sealed tube with DCM (60 mL) (does notcompletely dissolve, a brown suspension is obtained). The reactionmixture was cooled to −78° C. followed by the addition of granularaluminum chloride (2.84 g, 21.27 mmol). Ethylene was bubbled through thesolution for 7 min and the tube was sealed. A precipitate formed and thereaction mixture was stirred at −78° C. for 15 min and then allowed toreach room temperature. The reaction mixture was stirred for 2 h at roomtemperature and then depressurized. LCMS analysis showed disappearanceof starting material and appearance of the tetralone product. Thereaction mixture was poured over ice, diluted with DCM and stirred untilthe ice melted. The organic layer was washed with brine, dried andconcentrated under reduced pressure. Purification on silica gel afforded(5R,7R)-7-(6-oxo-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one(1.05 g, 3.68 mmol). HPLC retention time=0.74 min (Condition I); LC/MSM⁺¹=286; ¹H NMR (400 MHz, chloroform-d) δ 7.23-7.11 (m, 3H), 5.68 (br.s., 1H), 4.45-4.30 (m, 2H), 3.59 (s, 2H), 3.31-3.18 (m, 1H), 3.08 (t,J=6.8 Hz, 2H), 2.58 (t, J=6.7 Hz, 2H), 2.42-2.39 (m, 1H), 2.32-2.15 (m,2H), 2.09-1.99 (m, 1H), 1.91-1.83 (m, 1H), 1.82-1.72 (m, 1H).

Intermediate 8(1-Amino-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol

Intermediate 8A: 6-Iodo-3,4-dihydronaphthalen-1(2H)-one

To a stirred clear solution of 6-amino-3,4-dihydronaphthalen-1(2H)-one(15 g, 93 mmol) in acetic acid (150 mL) and water (150 mL) was addedsulfuric acid (5.5 mL, 101 mmol) dropwise at 0° C. A solution of sodiumnitrite (12.90 g, 187 mmol) in water (100 mL) was then added dropwiseover 40 min at the same temperature. The mixture was stirred at 0° C.for 10 min before being added to a stirred solution of sodium iodide(55.8 g, 372 mmol) in water (600 mL) slowly over 2 h at 0° C. Theresulting brown suspension was stirred at 0° C. for 30 min and at roomtemperature for 1 h. The mixture was extracted with ethyl acetate (400mL, 2×100 mL). The combined ethyl acetate extracts were washed withwater (60 mL), saturated aqueous Na₂S₂O₃ solution until the brown colordisappeared, and saturated aqueous K₃PO₄ (60 mL) solution, dried overanhydrous sodium sulfate, and concentrated under reduced pressure. Flashchromatography purification (330 g silica gel column, gradient elutionfrom 5 to 15% ethyl acetate in hexanes) afforded6-iodo-3,4-dihydronaphthalen-1(2H)-one (17.3 g, 63.6 mmol) as a solid.LC/MS M⁺¹=273.

Intermediate 8B:(S)—N-(6-Iodo-3,4-dihydronaphthalen-1(2H)-ylidene)-2-(methoxymethyl)pyrrolidin-1-amine

To a stirred mixture of 6-iodo-3,4-dihydronaphthalen-1(2H)-one (I-8A,20.90 g, 77 mmol), p-toluenesulfonic acid monohydrate (0.584 g, 3.07mmol), and cyclohexane (40 mL) was added(S)-2-(methoxymethyl)pyrrolidin-1-amine (10 g, 77 mmol) dropwise at roomtemperature under nitrogen. The mixture was heated with azeotropicremoval of water for 5 h. The reaction mixture was diluted with ethylacetate (20 mL) and mixed with saturated aqueous sodium bicarbonatesolution (15 mL). The aqueous layer was separated and extracted withethyl acetate (2×30 mL). The combined organic solutions were dried overanhydrous sodium sulfate and concentrated under reduced pressure. Flashchromatography purification (330 g silica gel column, gradient elutionfrom 0% to 20% EtOAc in hexanes) afforded(S)—N-(6-iodo-3,4-dihydronaphthalen-1(2H)-ylidene)-2-(methoxymethyl)pyrrolidin-1-amine(29.1 g, 76 mmol) as a yellow liquid. LC/MS M⁺¹=385.

Intermediate 8C: (R)-2-Hexyl-6-iodo-3,4-dihydronaphthalen-1(2H)-one

To a stirred solution of diisopropylamine (19.43 mL, 136 mmol) inanhydrous tetrahydrofuran (250 mL) was added butyl lithium solution (2.5M in hexanes, 39.4 mL, 98 mmol) dropwise at 0° C. under nitrogen. Theresulting solution was stirred at the same temperature for 15 min beforea solution of(S)—N-(6-iodo-3,4-dihydronaphthalen-1(2H)-ylidene)-2-(methoxymethyl)pyrrolidin-1-amine(I-8B, 29.1 g, 76 mmol) in anhydrous tetrahydrofuran (100 mL) was addeddropwise. The reaction solution was stirred at 0° C. for 2 h. A solutionof 1-iodohexane (22.35 mL, 151 mmol) in tetrahydrofuran (50 mL) wasadded dropwise at −78° C. and the mixture was stirred at the sametemperature for 2 h. The temperature was raised to room temperature over1.5 h. The reaction mixture was quenched with saturated aqueous ammoniumchloride solution (50 mL) and water (50 mL). The mixture was extractedwith hexanes (200 mL) and ethyl acetate (3×50 mL). The combined extractswere dried over anhydrous sodium sulfate and concentrated under reducedpressure to give an oil. The oil was dissolved in THF (200 mL). Asolution of cupric chloride, dihydrate (52 g) in water (220 mL) wasadded at 0° C. dropwise and the mixture was vigorously stirred at roomtemperature overnight. Aqueous ammonia solution was added to raise thepH to approximately 9. The mixture was extracted with hexane (100 mL)and diethyl ether (2×100 mL). The combined extracts were dried overanhydrous sodium sulfate and concentrated. Flash chromatographypurification (330 g silica gel column, gradient elution from 0 to 15%EtOAc in hexanes) afforded(R)-2-hexyl-6-iodo-3,4-dihydronaphthalen-1(2H)-one (21.6 g, 60.6 mmol)as a white solid containing some of the (S) isomer, which was removed inthe subsequent step. LC/MS M⁺¹=357.

Intermediate 8D: (R)-2-Hexyl-6-iodo-1,2,3,4-tetrahydronaphthalene

To a stirred solution of(R)-2-hexyl-6-iodo-3,4-dihydronaphthalen-1(2H)-one (I-8C, 21.6 g, 60.6mmol) in dichloromethane (10 mL) and 100% EtOH (100 mL) was added sodiumborohydride (4.59 g, 121 mmol) portionwise. The mixture was stirred atroom temperature for 2 h before being quenched by adding acetone slowly(cooled with water bath). The mixture was concentrated under reducedpressure. The residue was mixed with saturated aqueous ammonium chloridesolution (100 mL) and water (50 mL) and extracted with ethyl acetate(100 mL, 2×50 mL). The combined ethyl acetate extracts were dried overanhydrous sodium sulfate and concentrated under reduced pressure to givean oil. The oil was dissolved in triethylsilane (70 mL, 438 mmol). TFA(100 mL, 1298 mmol) was added with vigorous stirring. The mixture wasstirred at room temperature under nitrogen for 2.5 h. After water (150mL) was added, the mixture was extracted with hexanes (100 mL, 2×50 mL).The combined extracts were washed with water (50 mL) and then saturatedaqueous sodium bicarbonate solution (50 mL), dried over anhydrous sodiumsulfate, and concentrated to give a yellow liquid. Flash chromatographypurification (330 g silica gel column, gradient elution from 0 to 12%EtOAc in hexanes) afforded(R)-2-hexyl-6-iodo-1,2,3,4-tetrahydronaphthalene (18.4 g, 53.8 mmol) asa colorless liquid. ¹H NMR (400 MHz, chloroform-d) δ 7.41 (s, 1H), 7.38(dd, J=7.9, 1.8 Hz, 1H), 6.79 (d, J=8.1 Hz, 1H), 2.82-2.71 (m, 3H), 2.31(dd, J=16.5, 10.6 Hz, 1H), 1.93-1.85 (m, 1H), 1.72-1.60 (m, 1H),1.41-1.24 (m, 11H), 0.92-0.85 (m, 3H). Chiral SFC separation (CHIRALPAK®AS-H 25×3.0 cm, 5 μm, CO₂/MeOH=95/5, 180 mL/min, 230 nm) gave(R)-2-hexyl-6-iodo-1,2,3,4-tetrahydronaphthalene (11.8 g, PK2) and its(S)-isomer (1.4 g, PK1) as liquids.

Intermediate 8E:3-((R)-6-Hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentanone

Nitrogen gas was bubbled through a mixture of(R)-2-hexyl-6-iodo-1,2,3,4-tetrahydronaphthalene (I-8D, 11.8 g, 34.5mmol), tetrabutylammonium chloride (9.58 g, 34.5 mmol), potassiumacetate (10.15 g, 103 mmol), palladium (II) acetate (0.774 g, 3.45mmol), and anhydrous DMF (100 mL) for 3 min before cyclopent-2-enol (6.8g, 81 mmol, prepared according to Larock, R. C. et al., Tetrahedron,50(2):305-321 (1994)) was added. Nitrogen gas was bubbled through thesolution for an additional 2 min. The mixture was stirred at 80° C.under nitrogen for 2.5 h and then concentrated to remove DMF. Theresidue was mixed with water (150 ml) and extracted with ethyl acetate(4×50 mL). The combined ethyl acetate solutions were washed with water(30 mL), dried over anhydrous sodium sulfate, filtered through a silicagel pad, and concentrated under reduced pressure. Flash chromatographypurification (220 g silica gel column, gradient elution from 5 to 50%ethyl acetate in hexanes) afforded3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentanone (5.96 g,19.97 mmol) LC/MS M=299.

Intermediate 8F: Methyl1-amino-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentanecarboxylate

A mixture of3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentanone (I-8E,5.96 g, 19.97 mmol), ammonium chloride (5.34 g, 100 mmol), sodiumcyanide (4.89 g, 100 mmol), 7 M methanol solution of ammonia (28.5 ml,200 mmol), and dichloromethane (15 mL) was stirred at room temperaturefor 1 day. Additional 7 M methanol solution of ammonia (15 mL) wasadded. The mixture was stirred at room temperature for 1 day and thenconcentrated. The residue was partitioned between ethyl acetate (70 mL)and saturated aqueous sodium bicarbonate solution (100 mL). The aqueouslayer was separated and extracted with ethyl acetate (3×50 mL). Thecombined ethyl acetate solutions were dried over anhydrous sodiumsulfate and concentrated under reduced pressure to give1-amino-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentanecarbonitrileas a semi-solid. The semi-solid material was mixed with a mixture ofconcentrated hydrochloric acid (56 ml, 1843 mmol), water (28 ml, 1554mmol), acetic acid (35 ml), and dioxane (35 ml). The mixture was stirredat 100° C. under nitrogen for 10 h and then concentrated to give asolid. The solid was dissolved in methanol (20 mL) and mixed withtoluene (20 mL). The mixture was concentrated to dryness. This dryingprocedure was repeated one more time to give a dry solid. The solid wasdissolved in anhydrous methanol (300 mL). Thionyl chloride (11.66 mL,160 mmol) was added dropwise at 0° C. under nitrogen. The reactionmixture was stirred at 70° C. for 7 h before being concentrated. Theresidue was made basic with saturated aqueous sodium bicarbonatesolution (100 mL) and some potassium carbonate solid and extracted withethyl acetate (100 mL, 3×30 mL). The combined ethyl acetate extractswere dried over anhydrous sodium sulfate and concentrated under reducedpressure. Flash chromatography purification (80 g silica gel column,gradient elution from 20 to 100% ethyl acetate in hexanes) affordedmethyl1-amino-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentanecarboxylate(5.7 g, 15.94 mmol) as a liquid. LC/MS M⁺¹=358.

Intermediate 8

Methyl1-amino-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentanecarboxylate(I-8F, 5.7 g, 16 mmol) was dissolved in EtOH (60 mL) and methylenechloride (15 mL). Sodium borohydride (2.5 g, 67 mmol) was added. Themixture was stirred at room temperature overnight. Hydrochloric acid (6N aqueous, 40 mL) was added slowly at 0° C. to make pH approximately 1.After the mixture was stirred at room temperature for 60 min, sodiumhydroxide (10 g in 20 mL of water) was added to make pH approximately12. The mixture was stirred at room temperature for 40 min before beingconcentrated to remove organic solvents. The aqueous residue was dilutedwith water (20 mL) and extracted with EtOAc (100 mL, 2×50 mL). Thecombined ethyl acetate extracts were dried over anhydrous sodiumsulfate, decolored by charcoal, filtered through CELITE® pad, andconcentrated under reduced pressure to give(1-amino-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol(5.0 g, 15 mmol) as a white solid.

Examples 1 and 2 and Compounds 3 and 4

Intermediate 8 was separated into 3 fractions: F1 (Peak 1), F2 (Peak 2and Peak 3), F3 (Peak 4)) using chiral SFC (Column: CHIRALPAK® AD-H 25×3cm, 5 μm; Mobile Phase: CO₂/(MeOH+0.1% DEA)=88/12; Flow Rate: 200mL/min; Detector Wavelength: 220 nm; Column Temperature: 35° C.). Thesecond fraction (F2) was separated again using chiral SFC (Column:CHIRALPAK® AS-H 25×3 cm, 5 μm; Mobile Phase:CO₂/[MeOH-MeCN (1:1) +0.5%DEA]=88/12; Flow Rate: 180 mL/min; Detector Wavelength: 220 nm; ColumnTemperature: 35° C.) to give Peak 2 and Peak 3. All four isomers arewhite solids with LC/MS M⁺¹=330.

Example 1 Peak 2

¹H NMR (400 MHz, chloroform-d) δ 7.07-6.95 (m, 3H), 3.54-3.44 (m, 2H),3.34 (tt, J=11.0, 7.0 Hz, 1H), 2.92-2.74 (m, 3H), 2.39 (dd, J=16.4, 10.7Hz, 1H), 2.29-2.14 (m, 1H), 2.08-1.84 (m, 3H), 1.76-1.65 (m, 3H),1.46-1.26 (m, 12H), 0.99-0.85 (m, 3H).

Example 2 Peak 4

¹H NMR (400 MHz, chloroform-d) δ 7.06-6.92 (m, 3H), 3.52-3.37 (m, 2H),3.09-2.93 (m, 1H), 2.88-2.72 (m, 3H), 2.35 (dd, J=15.8, 10.6 Hz, 1H),2.26 (dd, J=12.7, 7.8 Hz, 1H), 2.11-2.00 (m, 1H), 1.97-1.84 (m, 2H),1.78-1.60 (m, 3H), 1.43-1.22 (m, 12H), 0.95-0.83 (m, 3H).

Compound 3 Peak 1

¹H NMR (400 MHz, chloroform-d) δ 7.03-6.92 (m, 3H), 3.46 (s, 2H),3.39-3.24 (m, 1H), 2.88-2.72 (m, 3H), 2.36 (dd, J=16.3, 10.8 Hz, 1H),2.27-2.13 (m, 1H), 2.08-1.82 (m, 3H), 1.76-1.63 (m, 3H), 1.43-1.20 (m,12H), 0.94-0.85 (m, 3H).

Compound 4 Peak 3

¹H NMR (400 MHz, chloroform-d) δ 7.02-6.92 (m, 3H), 3.51-3.37 (m, 2H),3.08-2.94 (m, 1H), 2.87-2.71 (m, 3H), 2.35 (dd, J=16.2, 11.1 Hz, 1H),2.25 (dd, J=13.1, 7.8 Hz, 1H), 2.12-1.97 (m, 1H), 1.97-1.83 (m, 2H),1.81-1.59 (m, 3H), 1.43-1.21 (m, 12H), 0.96-0.74 (m, 3H).

Compounds 5-8 were prepared according to the general synthesis andseparation procedures for Intermediate 8, Examples 1-2, and Compounds3-4 using (S)-enantiomer (PK1) of the iodide Intermediate I-8D. All fourisomers (Compounds 5-8) had MW=329.5; LC/MS M⁺¹=330; HPLC condition: C.

TABLE 1 Example HPLC No. Structure Name RT (min.) 1

((1R,3R)-1-amino-3-((R)-6-hexyl- 5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol 3.78 2

((1R,3S)-1-amino-3-((R)-6-hexyl- 5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol 3.78

TABLE 2 Compound HPLC Ret. No. Structure Name Time (min.) 3

((1S,3S)-1-amino-3-((R)-6- hexyl-5,6,7,8- tetrahydronaphthalen-2-yl)cyclopentyl)methanol 3.78 4

((1S,3R)-1-amino-3-((R)-6- hexyl-5,6,7,8- tetrahydronaphthalen-2-yl)cyclopentyl)methanol 3.78 5

((1S,3S)-1-amino-3-((S)-6- hexyl-5,6,7,8- tetrahydronaphthalen-2-yl)cyclopentyl)methanol 3.78 6

((1R,3R)-1-amino-3-((S)-6- hexyl-5,6,7,8- tetrahydronaphthalen-2-yl)cyclopentyl)methanol 3.78 7

((1S,3R)-1-amino-3-((S)-6- hexyl-5,6,7,8- tetrahydronaphthalen-2-yl)cyclopentyl)methanol 3.77 8

((1R,3S)-1-amino-3-((S)-6- hexyl-5,6,7,8- tetrahydronaphthalen-2-yl)cyclopentyl)methanol 3.77

Alternate Preparation of Example 2 Preparation 2A:(5R,7S)-7-(6-Hexyl-7,8-dihydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one

To a mixture of6-((5R,7S)-2-oxo-3-oxa-1-azaspiro[4.4]nonan-7-yl)-3,4-dihydronaphthalen-2-yltrifluoromethanesulfonate(Int. 6, 1 g, 2.396 mmol) and NMP (2.306 mL, 23.96 mmol) in THF (20 mL)at −40° C. was added a solution of lithium bis(trimethylsilyl)amide inTHF (2.396 mL, 2.396 mmol). The reaction mixture was stirred for 15minutes, and then ferric acetylacetonate (0.042 g, 0.120 mmol) andhexylmagnesium bromide in ether (2.396 mL, 4.79 mmol) were added. Thereaction mixture was stirred for 30 minutes and quenched with water. Thereaction mixture was diluted with ethyl acetate and washed with 1M HCl.The organic layer was dried with MgSO₄, filtered and concentrated. Thecrude material was purified on a silica gel cartridge (40 g) using anEtOAc/hexane gradient (0-100% EtOAc over 12 CV) to afford 662 mg of(5R,7S)-7-(6-hexyl-7,8-dihydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-oneas a white solid. HPLC retention time=1.28 min (Condition G); LC/MSM⁺¹=354: ¹H NMR (400 MHz, chloroform-d) δ 7.03-6.88 (m, 3H), 6.21 (s,1H), 5.16 (br. s., 1H), 4.44-4.15 (m, 2H), 3.13-2.96 (m, 1H), 2.80 (t,J=8.0 Hz, 2H), 2.47-2.08 (m, 6H), 2.05-1.93 (m, 2H), 1.90-1.73 (m, 1H),1.68-1.43 (m, 4H), 1.41-1.22 (m, 5H), 1.01-0.83 (m, 3H).

Preparation 2B:(5R,7S)-7-((R)-6-Hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one

A mixture of(5R,7S)-7-(6-hexyl-7,8-dihydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one(2A, 760 mg, 2.15 mmol) and(−)-2,3-bis[(2R,5R)-2,5-dimethylphospholanyl]-N-[3,5-bis(trifluoromethyl)phenyl]maleicimide(1,5-cyclooctadiene)rhodium(I) tetrafluoroborate (273 mg, 0.43 mmol) inMeOH (27 mL) was hydrogenated at 850 PSI for 1000 minutes using a 100 mLHEL autoclave. The reaction mixture was filtered and concentrated invacuo. The crude material was purified on a silica gel cartridge (40 g)using an EtOAc/hexane gradient (0-100% EtOAc over 20 minutes) to afford640 mg of(5R,7S)-7-(6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-onein a 1:2 ratio of the two isomers. The desired major isomer wasseparated using a CHIRALPAK® AS-H column under SFC conditions (35% MeOHin CO₂). Retention time=5.14 min. Recovered 400 mg of(5R,7S)-7-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one.¹H NMR (400 MHz, methanol-d₄) δ 7.02-6.91 (m, 3H), 4.47-4.20 (m, 2H),3.02 (tt, J=11.0, 7.2 Hz, 1H), 2.87-2.74 (m, 3H), 2.41-2.24 (m, 2H),2.17-2.03 (m, 2H), 2.00-1.89 (m, 3H), 1.86-1.74 (m, 1H), 1.73-1.61 (m,1H), 1.51-1.28 (m, 11H), 0.99-0.88 (m, 3H).

Alternate Preparation of Preparation 2B

A mixture of(5R,7S)-7-(6-hexyl-7,8-dihydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one(2A, 2.1 g, 5.94 mmol) in DCM (10 ml) was added dropwise to a solutionof ((1R,2S,3R,5R)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-yl)borane in DCM(30 ml) at −35° C. under nitrogen. (The borane reagent was made asfollowing: To a mixture of S-Alpine-Boramine (8.4 g, 20.18 mmol) in THF(35 ml) was added BF₃ ethereate (5.11 ml, 40.4 mmol) at roomtemperature. The mixture was stirred at room temperature for 1.5 h,filtered under nitrogen, and the cake was washed with cold THF (2×6 ml).The filtrate and washes were combined and concentrated under vacuum. Tothe residue was added DCM (20 ml) and the solution was concentratedagain. The reagent obtained was redissolved in DCM (30 ml) and useddirectly for the hydroboration step). After stirring at −35° C. to −30°C. for 4 h and at −25° C. to −20° C. for 2 h, MeOH (3.6 mL) was addedand the reaction mixture was stirred at −10° C. for 10 min and at 0° C.for 10 min. The mixture was diluted with THF (25 ml), then a solution ofNaOH (6N, 9.9 ml, 59.4 mmol) was added dropwise followed by H₂O₂ (30%,6.07 ml, 59.4 mmol) and the mixture was stirred at room temperature for16 h. To the mixture was added DCM (100 ml) and water (50 ml). The wholesolution was filtered through a pad of CELITE® and the cake was washedwith DCM. The two layers were separated, the aqueous layer was extractedwith DCM (50 ml) which was combined with the organic layer. The combinedorganic layers were washed with water (100 ml) and brine (100 ml), driedover anhydrous Na₂SO₄ and concentrated in vacuo. The crude material waspurified on a silica gel cartridge (40 g) using an EtOAc/hexane gradient(0-50% EtOAc over 55 minutes) to afford 1.9 g (86%) of(5R,7S)-7-(6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-onein a 8:1 ratio of two isomers. The mixture was carried into the nextstep without separating the diastereomers.

To(5R,7S)-7-(6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one(3.2 g, 8.61 mmol) in MeOH (30 ml) was added Pd/C (10%, 1.1 g). A slightvacuum was applied to the reaction flask, followed by back filling withhydrogen from a hydrogen balloon. After stirring at room temperature for6 h, EtOAc (20 ml) was added to dissolve the precipitate. A slightvacuum was applied to the reaction flask, followed by back filling withhydrogen from a hydrogen balloon and the contents stirred at roomtemperature for 16 h. The mixture was filtered through a pad of CELITE®and the cake was washed with EtOAc, DCM, MeOH and EtOAc. The combinedsolvents were concentrated in vacuo to afford the crude mixture (3.06 g)as an 8:1 diastereomeric mixture. The major isomer was separated using aCHIRALPAK® AS-H column under SFC conditions (35% MeOH in CO₂). Retentiontime=4.64 min. Recovered 2.35 g (77%) of(5R,7S)-7-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one.

Example 2

To a mixture of(5R,7S)-7-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one(400 mg, 1.125 mmol) in dioxane (30 mL) was added aqueous NaOH (1N, 20mL). The reaction mixture was heated at 100° C. for 3 days, cooled toroom temperature, diluted with ethyl acetate and washed with water. Theorganic layer was dried with MgSO₄, filtered and concentrated. The crudematerial was purified on a silica gel cartridge (24 g) using an 20% (2NNH₃/MeOH) in DCM/DCM gradient (0-75% of 20% (2N NH₃/MeOH) in DCM over 13CV) to afford 290 mg of((1R,3S)-1-amino-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol.HPLC retention time=10.09 min (Condition H); LC/MS M⁺¹=330; ¹H NMR (400MHz, methanol-d₄) δ 7.03-6.91 (m, 3H), 3.54-3.41 (m, 2H), 3.01 (tt,J=11.1, 7.2 Hz, 1H), 2.87-2.69 (m, 3H), 2.34 (dd, J=16.2, 10.5 Hz, 1H),2.20 (dd, J=13.0, 7.5 Hz, 1H), 2.07-1.84 (m, 3H), 1.83-1.60 (m, 3H),1.60-1.48 (m, 1H), 1.47-1.25 (m, 11H), 1.00-0.88 (m, 3H).

Example 2 Free Base, Form N-1

Example 2, free base, Form N-1 was obtained by preparing a stocksolution containing 385 mg of Example 2 dissolved in a mixture of 18 mlTHF and 1.25 ml H₂O (20 mg/ml). Next, 52 μl of the stock solution wasevaporated to dryness. To the dried material was added 52 μl of a 50:50ratio ethanol:heptane solution. The resulting solution was evaporated toyield plates of crystalline material.

Example 2 Mono-HCl Salt, Monohydrate, Form H-1

The monohydrate, mono-HCl salt of Example 2, Form H-1 was obtained bypreparing a 50:50 aqueous methanol (200 μl) solution containing 3.2 mgof Example 2. Next, 400 μl of a 0.025M aqueous HCl solution was addeddropwise with stirring. The resulting solution was evaporated to yieldplates of crystalline material.

Example 2 Mono-HCl Salt, Monohydrate, Form H-2

The monohydrate, mono-HCl salt of Example 2, Form H-2 was obtained bypreparing a 50:50 aqueous THF (200 μl) solution containing 3.2 mg ofExample 2. Next, 400 μl of a 0.025M aqueous HCl solution was addeddropwise with stirring. The solution was then evaporated to yield platesof the monohydrate H-2 form of the mono-HCl salt of Example 2.

Example 2 Mono-HCl Salt, Form N-3

The mono-HCl salt of Example 2, Form N-3 was obtained by preparing a 200μl solution of isopropyl alcohol containing 3.2 mg of Example 2. Next, a400 μl of a 0.025 M alcoholic HCl solution was added dropwise withstirring. The solution was evaporated to yield plates of the N-3 form ofthe mono-HCl salt of Example 2.

Example 2 Mono-HCl Salt, Form N-4

The mono-HCl salt of Example 2, Form N-3 was obtained by preparing a 200μl of a 50:50 methanol/THF solution containing 3.2 mg of Example 2.Next, 400 μl of a 0.025 M alcoholic HCl solution was added dropwise withstirring. The resulting solution was evaporated to yield plates of theN-4 form of the mono-HCl salt of Example 2.

Example 2 Monohydrate, Hemi-L-Malic Acid Salt, Form H-1

The monohydrate, hemi-L-malic acid salt, Form H-1 was obtained bypreparing a 200 μl 50:50 aqueous THF solution containing 3.2 mg ofExample 2. Next, 240 μl of a 0.042 M alcoholic L-malic acid solution wasadded dropwise with stirring. The resulting solution was evaporated toyield plates of the H-1 form of the hemi-L-malic acid salt of Example 2.

Example 2 Monohydrate, Hemi-Malonic Acid Salt, Form H-1

The monohydrate, hemi-malonic acid salt, Form H-1 was obtained bypreparing a 200 μl 50:50 aqueous THF solution containing 3.2 mg ofExample 2. Next, 193 μl of a 0.052 M alcoholic malonic acid solution wasadded dropwise with stirring. The resulting solution was evaporated toyield plates of the H-1 form of the hemi-malonic acid salt of Example 2.

Example 2 ⅓-Hydrate, Phosphoric Acid Salt, Form H.33-1

The ⅓-hydrate, phosphoric acid salt, Form H.33-1 was obtained bypreparing a 200 μl 50:50 aqueous methanol solution containing 3.2 mg ofExample 2. Next, 136 μl of a 0.073 M alcoholic phosphoric acid solutionwas added dropwise with stirring. The resulting solution was evaporatedto yield plates of the H.33-1 form of the phosphoric acid salt ofExample 2.

Example 2 R-(+)-Mandelic Acid Salt, Form N-1

The R-(+)-mandelic acid salt, Form N-1 was prepared by adding Example 2and an equimolar amount of R-(+)-mandelic acid to a mixture of methanoland acetonitrile. The solution was evaporated to yield plates of the N-1form of the mono-R-(+)-mandelic acid salt of Example 2.

Example 9((1R,3R)-1-Amino-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methyldihydrogen phosphate

To a stirred solution of((1R,3R)-1-amino-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol(Example 1, 10 mg, 0.030 mmol) in anhydrous acetonitrile (1 mL) at 0° C.was added pyrophosphoryl chloride (0.042 mL, 0.303 mmol). The clearsolution obtained was stirred at the same temperature for 5 min and atRT overnight. After water (0.4 mL) was added, the mixture was stirred atRT for 1 h. Purification using reverse phase HPLC (Luna Axia 5μ c1830×100 mm, 10 min. gradient from 40% to 100% of Solvent B, Solvent A:0.1% TFA in water, Solvent B: 0.1% TFA in MeCN), concentration andlyophilization gave((1R,3R)-1-amino-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methyldihydrogen phosphate (2 mg, 4.15 μmol, 13.68% yield) as a white solid.HPLC retention time=3.94 min (Condition C); LC/MS M⁺¹=410; ¹H NMR (500MHz, methanol-d₄+KOH) δ 6.97-6.87 (m, 3H), 3.77-3.71 (m, 1H), 3.71-3.66(m, 1H), 2.83-2.70 (m, 3H), 2.32 (dd, J=16.2, 10.7 Hz, 1H), 2.14-1.98(m, 2H), 1.96-1.83 (m, 2H), 1.74-1.60 (m, 3H), 1.59-1.49 (m, 1H),1.45-1.26 (m, 11H), 0.94-0.87 (m, 3H), one proton under methanol solventpeak (˜3.3 ppm).

Example 10((1R,3S)-1-Amino-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methyldihydrogen phosphate

A mixture of phosphorus pentoxide (150 mg, 0.528 mmol) and 85%phosphoric acid (0.15 mL, 10.01 μmol) was stirred at 100° C. undernitrogen for 1 h before((1R,3S)-1-amino-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol(Example 1, 6 mg, 0.018 mmol) was added. The solution was stirred at thesame temperature for 3 h. Water (0.5 mL) was added at room temperature.The mixture was stirred at room temperature for 1 h. Purification usingreverse phase HPLC (PHENOMENEX® Luna Axia 5μ c18 30×100 mm, 10 minuterun, Solvent A: 10% MeOH: 90% H₂O: 0.1% TFA, Solvent B: 90% MeOH, 10%H₂O, 0.1% TFA), concentration, and lyophilization gave((1R,3S)-1-amino-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methyldihydrogen phosphate (4 mg, 9.38 μmol) as a white solid. LC/MS M⁺¹=410.HPLC retention time=3.96 min (Condition C). ¹H NMR (400 MHz,methanol-d₄+CDCl₃) δ 7.10-6.83 (m, 3H), 4.06-3.77 (m, 2H), 3.20-3.06 (m,1H), 2.85-2.74 (m, 2H), 2.48 (dd, J=12.9, 6.9 Hz, 1H), 2.40-2.28 (m,1H), 2.19-2.08 (m, 1H), 2.05-1.90 (m, 4H), 1.80-1.62 (m, 2H), 1.48-1.22(m, 12H), 0.95-0.86 (m, 3H).

Alternate Preparation of Example 10 Alternate Preparation 10A:tert-Butyl((1R,3S)-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)-1-(hydroxymethyl)cyclopentyl)carbamate

To a stirred solution of((1R,3S)-1-amino-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol(Example 2, 270 mg, 0.819 mmol) in anhydrous dichloromethane (6 mL) wasadded di-tert-butyl dicarbonate (536 mg, 2.458 mmol). The resultingsolution was stirred at room temperature for 3 h. The mixture wasconcentrated. Flash chromatography purification (24 g silica gel column,gradient elution from 10 to 60% of ethyl acetate in hexanes) affordedtert-butyl((1R,3S)-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)-1-(hydroxymethyl)cyclopentyl)carbamate(337 mg, 0.784 mmol, 96% yield) as a white solid. LC/MS M⁺¹=430.

Alternate Preparation 10B:tert-Butyl((1R,3S)-1-(((bis(2-(trimethylsilyl)ethoxy)phosphoryl)oxy)methyl)-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)carbamate

To a stirred solution oftert-butyl((1R,3S)-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)-1-(hydroxymethyl)cyclopentyl)carbamate(Preparation 10A, 336 mg, 0.782 mmol) in anhydrous methylene chloride (7mL) was added bis(2-(trimethylsilyl)ethyl)diisopropylphosphoramidite(858 mg, 2.346 mmol) in one portion at 0° C. under nitrogen. Next, 1, 2,4-1H-triazole (162 mg, 2.346 mmol) was then added. The reaction mixturewas stirred at 40° C. for 18 h. The solution was cooled to 0° C. beforehydrogen peroxide (0.781 mL, 7.82 mmol) was added. The mixture wasstirred at 0° C. for 30 minutes at room temperature for 1 h beforemethanol (3 mL) was added to make the mixture a homogeneous solution.The solution was stirred at room temperature for 1 h. A saturatedaqueous sodium thiosulfate solution (5 mL) was added to quench thereaction. The mixture was concentrated under reduced pressure andextracted with ethyl acetate (3×4 mL). The combined organic solutionswere dried (Na₂SO₄) and concentrated under reduced pressure. Flashchromatography purification (24 g silica gel column, gradient elutionfrom 5 to 25% of ethyl acetate in hexanes) affordedtert-butyl((1R,3S)-1-(((bis(2-(trimethylsilyl)ethoxy)phosphoryl)oxy)methyl)-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)carbamate(514 mg, 0.724 mmol) as a liquid.

Example 10

To a stirred solution oftert-butyl((1R,3S)-1-(((bis(2-(trimethylsilyl)ethoxy)phosphoryl)oxy)methyl)-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)carbamate (Preparation 10B, 500 mg, 0.704 mmol) in dichloromethane (6mL) was added TFA (6 mL) slowly at 0° C. The mixture was stirred at roomtemperature for 3 h before 90 mL of heptanes was added. The solution wasconcentrated under reduced pressure. Next, 70 mL of methanol was addedto the solid residue, followed by 1N aq NaOH (4 mL). HOAc (0.4 mL) wasthen added at 60° C. to acidify the solution to pH=4. The solid-liquidmixture was stirred at 60° C. for 1 h. The solid was separated, washedwith methanol, water, methanol, ethyl acetate, and methanol.Lyophilization gave((1R,3S)-1-amino-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methyldihydrogen phosphate (257 mg, 0.619 mmol, 88% yield) as a white solid.LC/MS M⁺¹=410; ¹H NMR (400 MHz, methanol-d₄) (+KOH) δ 7.00-6.86 (m, 3H),3.78-3.62 (m, 2H), 3.09-2.97 (m, 1H), 2.84-2.67 (m, 3H), 2.38-2.22 (m,2H), 2.03-1.74 (m, 4H), 1.73-1.60 (m, 2H), 1.50 (t, J=12.3 Hz, 1H),1.44-1.27 (m, 11H), 0.95-0.86 (m, 3H).

Comparative Compound 11(1R,3R)-1-Amino-3-(6-(pentyloxy)naphthalen-2-yl)cyclopentyl)methanol

Comparative Compound 11 was disclosed in WO 2008/079382, Example Q.1.

Intermediate 11A:(5R,7R)-7-(6-(Pentyloxy)naphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one

A mixture of 1-pentanol (6.13 mL, 56.4 mmol), p-toluenesulfonic acidmonohydrate (4.60 mg, 0.024 mmol), and trimethoxymethane (0.353 mL, 3.22mmol) was stirred at 100° C. for 3 hr with a slow air stream flowingover the mixture to remove methanol and some pentanol. The obtainedresidual liquid was mixed with(5R,7R)-7-(6-oxo-5,6,7,8-tetrahydronaphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one(Int. 7, 230 mg, 0.806 mmol) and stirred at 100° C. under nitrogen for2.5 hr. The solution was allowed to cool down to room temperature beforepalladium on carbon (172 mg, 0.081 mmol) was added, followed by ethylacetate (4 mL). The mixture was left to stir under a balloon-pressure ofhydrogen at room temperature overnight. The resulting mixtures werefiltered through a membrane filter and the filtrate was concentrated.Flash chromatography purification (24 g silica gel column, 0% to 70%ethyl acetate in hexanes) afforded 180 mg of material that requiredadditional purification. Supercritical Fluid Chromatographic separationafforded a major fraction by UV analysis identified as(5R,7R)-7-(6-(pentyloxy)naphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one(36 mg) as a solid. Instrument: Thar 350 Thar Analytical SFC-MS;Conditions: Analytical Conditions: Analytical Column: AD-H (0.46×25 cm,5 μm); BPR pressure: 100 bars; Temperature: 45° C.; Flow rate: 3.0mL/min; Mobile Phase: CO₂/MeOH (70/30); Detector Wavelength: UV 200-400nm. Preparative Conditions: Preparative Column: AD-H (3×25 cm, 5 μm);BPR pressure: 100 bars; Temperature: 35° C.; Flow rate: 120 mL/min;Mobile Phase: CO₂/MeOH (70/30); Detector Wavelength: 220 nm; Separationprogram: Stack injection; Injection: 2.5 mL with cycle time 480 sec.(Analytical SFC ret. time=11.68 min, purity >99.5%) HPLC retentiontime=1.11 min (Condition G); LC/MS M⁺¹=354. ¹H NMR (400 MHz,chloroform-d) δ 7.68 (d, J=8.4 Hz, 2H), 7.55 (s, 1H), 7.30 (s, 1H),7.21-7.04 (m, 2H), 6.48 (br. s., 1H), 4.50-4.28 (m, 2H), 4.07 (t, J=6.6Hz, 2H), 3.49-3.31 (m, 1H), 2.46 (dd, J=13.3, 7.6 Hz, 1H), 2.39-2.24 (m,1H), 2.24-2.12 (m, 1H), 2.12-2.00 (m, 1H), 2.00-1.90 (m, 1H), 1.90-1.76(m, 3H), 1.58-1.30 (m, 4H), 0.96 (t, J=7.0 Hz, 3H).

Comparative Compound 11:

To a solution of(5R,7R)-7-(6-(pentyloxy)naphthalen-2-yl)-3-oxa-1-azaspiro[4.4]nonan-2-one(36 mg, 0.102 mmol) in dioxane (2 mL) and water (0.8 mL) was added LiOH(36.6 mg, 1.528 mmol). The solution was heated to 90° C. and allowed tostir for 15 hours. The reaction mixture was cooled to room temperatureand was poured into ethyl acetate and washed with water. The crudematerial was then purified on reverse phase HPLC [Column: Luna Axia30*100 mm; Gradient time: 10 min; Flow rate=40 ml/min; Solvent A=10%MeOH-90% Water-0.1% TFA; Solvent B=90% MeOH-10% water-0.1% TFA; Start %B=20; Final % B=100]. The product containing fractions were collectedand dried under high vacuum to provide((1R,3R)-1-amino-3-(6-(pentyloxy)naphthalen-2-yl)cyclopentyl)methanol,TFA (31 mg) as a solid. HPLC retention time=0.90 min (Condition G);LC/MS M⁺¹=328. ¹H NMR (400 MHz, methanol-d₄) δ 7.75-7.66 (m, 2H),7.66-7.59 (m, 1H), 7.40-7.33 (m, 1H), 7.17 (d, J=2.6 Hz, 1H), 7.14-7.08(m, 1H), 4.07 (t, J=6.5 Hz, 2H), 3.74-3.60 (m, 2H), 3.59-3.41 (m, 1H),2.39-2.22 (m, 3H), 2.04-1.80 (m, 5H), 1.55-1.34 (m, 4H), 1.01-0.89 (m,3H).

Biological Assays

Mouse Whole Blood Phosphorylation (WBP) Assay

The compounds of Formula (III) require bioactivation throughphosphorylation of the alcohol to provide an active phosphate estercompound of Formula (II). According to Brinkmann, V. et al., (J. Biol.Chem. 277:21453-21457 (2002)), the stereoisomeric configuration of theamine bearing carbon center can influence the relative extent to whichthis phosphorylation takes place.

The relative extent of phospholylation of Examples 1-2 and Compounds 3-8were evaluated by incubation of the alcohol compounds in whole bloodfrom a mouse. The appearance of the phosphorylated compound was measuredafter 4 hours to determine the relative extent of phosphate esterformation. Whole blood was freshly obtained from BALB/C mice byretro-orbital bleeding and collected into EDTA containing tubes. TheEDTA treated whole blood was aliquoted into 1.4 mL polypropylene tubesin 96 well format (100 μL per sample) and spiked with test compound (1mM in DMSO) for a final concentration of 10 μM (n=2 per compound). Tubeswere sealed and vortexed then transferred to orbital shaker forincubation at 37° C., 225 RPM for 4 hours. At the end of the incubation,samples were spotted onto Ahlstrom 226 untreated Specimen CollectionPaper (25 μL per spot n=2) and allowed to air dry overnight. Dried BloodSpot (DBS) Cards were stored at ambient temperature in sealed plasticbags with desiccant added. When ready for analysis, a 6 mm punch(equivalent to 12.5 μl of wet blood) was taken at n=1 and placed in ashallow 96-well filter plate. Next, 105 μL of a mixture of 75%acetonitrile and 25% water containing Internal Standard was added andgently vortexed for 30 min then centrifuged. Supernatant was separatedfrom the protein pellet and 5 μl was injected. The parent (alcohol) andactive phosphate ester compounds were quantitatively analyzed, using aDBS calibration curve, by LC/MS/MS on a Triple Quadrapole Instrument.The area ratios of the phosphorylated compound to the parent (alcohol)compound were determined. A larger value for the ratio of thephosphorylated compound to the parent (alcohol) compound indicatedgreater phosphate ester compound formation from the parent (alcohol)compound. Table 2 shows the results (average of two experiments) forExamples 1-2 and Compounds 3-8 at 4 hours. For Examples 1-2 and Compound6, the area ratios of the phosphate ester compound formation from theparent (alcohol) compounds at 4 hour were at least 0.59. In contrast,the area ratios of the phosphate ester compound formation from parent(alcohol) compounds for Compounds 3-5 and 7-8 were 0.17 or less. In thisstudy, Examples 1-2 and Compound 6 were found to undergo phosphorylationto a greater extent than Compounds 3-5 and 7-8.

TABLE 2 Mouse Whole Blood Phosphorylation - Extent of PhosphorylationExample or Mouse Whole Blood Compound No. Phosphate Area Ratio at 4 hr 11.60 2 0.59 3 0.07 4 0.14 5 0.03 6 0.60 7 0.02 8 0.17In Vivo Phosphate Ester Formation in Mice

BALB/c mice were dosed orally with Example 1, Example 2, Compound 3, andCompound 4 (10 mg/kg as a solution or suspension in the vehicle,polyethylene glycol 300, “PEG300”). Blood was drawn at 24 hr, spottedonto Dried Blood Spot (DBS) Cards, and analyzed as described for the WBPassay. Reference material (parent alcohol and phosphate ester) wasanalyzed to optimize the LC-MS/MS assay and enable data reporting inconcentrations. DBS standard curves containing both parent alcohol andphosphate ester compounds were prepared and analyzed in the same manneras the study samples and analyzed by the optimized LC-MS/MS to quantifythe amount of phosphate ester compound formed. The results in Table 3represent the average results of all animals within each treatment group(n=3). A larger value for the phosphate ester compound concentrationindicated greater phosphate ester compound formation from parent(alcohol) compound. In this study, Examples 1-2 were found to undergophosphate ester formation to a greater extent than Compounds 3-4. Theresults of this in vivo study are consistent with the results obtainedin the above Mouse Whole Blood Phosphorylation study.

TABLE 3 In Vivo Phosphate Formation in Mice Example or Dosage Phosphateat 24 hr Compound No. (mg/kg) (nM) 1 10 475 2 10 202 3 10 14 4 10 46S1P₁ Binding Assay

Membranes were prepared from CHO cells expressing human S1P₁. Cellspellets (1×10⁹ cells/pellet) were suspended in buffer containing 20 mMHEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.5, 50mM NaCl, 2 mM EDTA (Ethylenediaminetetraacetic acid) and ProteaseInhibitor cocktail (Roche), and disrupted on ice using the Polytronhomogenizer. The homogenate was centrifuged at 20,000 rpm (48,000 g) andthe supernatant was discarded. The membrane pellets were resuspended inbuffer containing 50 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM MgCl₂, 2 mMEDTA and stored in aliquots at −80° C. after protein concentrationdetermination.

Membranes (2 μg/well) and 0.03 nM final concentration of ³³P-S1P ligand(1 mCi/ml, Perkin Elmer or American Radiolabeled Chemicals) diluted inassay buffer (50 mM HEPES, pH7.4, 5 mM MgCl₂, 1 mM CaCl₂, 0.5% fattyacid free BSA (bovine serum albumin), 1 mM NaF) were added to thecompound plates (384 FALCON® v-bottom plate (0.5 μl/well in a 11 point,3-fold dilution). Binding was performed for 45 minutes at roomtemperature, terminated by collecting the membranes onto 384-wellMillipore FB filter plates, and radioactivity was measured by TOPCOUNT®.The competition data of the test compounds over a range ofconcentrations was plotted as percentage inhibition of radioligandspecific binding. The IC₅₀ is defined as the concentration of competingligand needed to reduce specific binding by 50%. The IC₅₀ for Example 10was determined to be 0.01 nM.

Receptor [³⁵S] GTPγS Binding Assays

Compounds were loaded in a 384 FALCON® v-bottom plate (0.5 μl/well in a11 point, 3-fold dilution). Membranes prepared from S1P₁/CHO cells orEDG3-Ga15-bla HEK293T cells (EDG3 equivalent S1P₃) were added to thecompound plate (40 μl/well, final protein 3 μg/well) with MULTIDROP®.[³⁵S]GTP (1250 Ci/mmol, Perkin Elmer) was diluted in assay buffer: 20 mMHEPES, pH7.5, 10 mM MgCl₂, 150 mM NaCl, 1 mM EGTA (ethylene glycoltetraacetic acid), 1 mM DTT (Dithiothreitol), 10 μM GDP, 0.1% fatty acidfree BSA, and 10 μg/ml Saponin to 0.4 nM. 40 μl of the [³⁵S] GTPsolution was added to the compound plate with a final concentration of0.2 nM. The reaction was kept at room temperature for 45 min. At the endof incubation, all the mixtures in the compound plate were transferredto Millipore 384-well FB filter plates via the VELOCITY11® Vprep liquidhandler. The filter plate was washed with water 4 times by using themanifold Embla plate washer and dried at 60° C. for 45 min. MicroScint20 scintillation fluid (30 μl) was added to each well for counting onthe Packard TOPCOUNT®. EC₅₀ is defined as the agonist concentration thatcorresponds to 50% of the Ymax (maximal response) obtained for eachindividual compound tested. The EC₅₀ for Example 10 was determined to be0.9 nM in the assay utilizing membranes prepared from S1P₁/CHO cells.The EC₅₀ for Example 10 was determined to be >62,500 nM in the assayutilizing membranes prepared from EDG3-Ga15-bla HEK293T cells.

A smaller value for GTPγS S1P₁ EC₅₀ value indicated greater activity forthe compound in the GTPγS S1P₁ binding assay. A larger value for theGTPγS S1P₃ EC₅₀ value indicated less activity in the GTPγS S1P₃ bindingassay. Example 10, which is the active phosphate ester of Example 2,possessed activity as an agonist of S1P₁ and is selective over S1P₃.Thus the compounds of the present invention, which include Examples 1-2and 9-10 may be used in treating, preventing, or curing various S1P₁receptor-related conditions while reducing or minimizing the sideeffects due to S1P₃ activity. The surprising selectivity of thecompounds of the present invention indicate their potential use intreating, preventing, or curing autoimmune and inflammatory diseasessuch as multiple sclerosis, rheumatoid arthritis, inflammatory boweldisease, lupus, or psoriasis, while reducing or minimizing possible sideeffects due to S1P₃ activity. Other potential uses of the compounds ofthe present invention include minimizing or reducing rejection oftransplanted organs, while reducing or minimizing side effects due toS1P₃ activity.

S1P1 Receptor Internalization Assay

CHO-K1 cells expressing a GFP-tagged S1P1 receptor were plated in384-well poly-D-lysine coated tissue culture plates at 4×10³ cells/wellin 50 μl assay media (F12 with L-glutamine, 10% charcoal/dextran-treatedFBS, 1× penicillin-streptomycin, 1M HEPES). Cell plates were incubatedovernight at 37° C./5% CO₂. Test compound were introduced to the cellplate from a compound source plate at 11 point, 3 fold serial dilutionsand then the assay plates were incubated at 37° C./5% CO₂ for 45 min.Cells were fixed and stained with 6% formaldehyde and 15 μg/ml Hoechstdye in PBS (Ca²⁺/Mg²⁺ free) at room temperature for 15 minutes. Cellplates were washed 4 times with PBS (Ca²⁺/Mg²⁺ free) with addition of 50μl/PBS prior to plate sealing. Images were acquired by the CellomicsARRAYSCAN® VTI high content imager. Data analysis for EC₅₀ determinationrelative to internal control compound was accomplished using theCompartmental Analysis BioApplication on the Array Scan. The EC₅₀ isdefined as the agonist concentration that corresponds to 50% of the Ymax(maximal response) obtained for each individual compound tested and wasquantified using the 4 parameter logistic equation to fit the data. TheEC₅₀ for Example 9 was determined to be 361 nM in the assay

Blood Lymphocyte Reduction (BLR) Assay in Rodent

Lewis rats were dosed orally with vehicle alone (polyethylene glycol300, “PEG300”) or with2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol, hydrochloride (CAS:162359-56-0) as a solution in the vehicle at doses of 0.1 mg/kg, 0.5mg/kg and 3.0 mg/kg adjusted to reflect the free amount of test article.The results are provided in Table 4a and the level of lymphocytereduction at 24 hours post-dose was maximal at 3.0 mg/kg. The percentreduction in lymphocytes is dose-related but the relationship is notlinear, with non-proportional increases in dose being required to elicitsequentially greater reductions in lymphocyte counts. For example, inthis study to demonstrate a change of 13% (from 69% reduction to 82%reduction) required escalation of five-fold in dose (from 0.1 mg/kg to0.5 mg/kg). Furthermore, to demonstrate an additional change of 7% inthis study (from 82% reduction to 89% reduction) required escalation ofsix-fold in dose (from 0.5 mg/kg to 3.0 mg/kg). BALB/c mice were dosedorally with vehicle alone (polyethylene glycol 300, “PEG300”) or withExample 1, Example 2, Compound 6, Compound 8, or Comparative Compound11. Compounds were dosed as a solution or suspension in the vehicle,adjusted to reflect the free amount of test article in the event thatsalt forms are utilized. Blood was drawn at 24 hr and blood lymphocytecounts were determined on an ADVIA® 120 Hematology Analyzer (SiemensHealthcare Diagnostics). The results were measured as a reduction in thepercentage of circulating lymphocytes as compared to the vehicle treatedgroup at the time of measurement. The results represent the averageresults of all animals within each treatment group (n=2-4). The resultsof the Blood Lymphocyte Reduction assay (BLR) in mouse describedhereinabove are shown in Table 4b.

TABLE 4a 2-Amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol,hydrochloride Rat Blood Lymphocyte Reduction Assay at 24 hr post-doseDosage Compound Vehicle Control Percent reduction (mg/kg) Mean SEM MeanSEM vs. control 0.1 2.78 0.17 9.01 0.19 69% 0.5 1.64 0.25 9.01 0.19 82%3.0 1.02 0.06 9.01 0.19 89%

TABLE 4b Mouse Blood Lymphocyte Reduction Assay at 24 hr post-dosePercent Example or Dosage Compound Vehicle Control reduction Compound No(mg/kg) Mean SEM Mean SEM vs. control 1 1 0.7 0.11 5.39 0.67 88% 2 10.54 0.20 5.39 0.67 90% 6 1 1.2 0.21 5.9 1.75 78% 8 1 2.4 0.35 5.9 1.7559% 11 1 2.18 0.63 4.58 0.14 52%Pulmonary Toxicity Assay

The analysis of protein levels in bronchoalveolar lavage (BAL) fluidobtained from an animal were used to gauge pulmonary side effects.Increased levels of protein in BAL fluid were indicative of undesiredpulmonary effects, such as pulmonary edema. Example 1, Example 2,Compound 6, Compound 8, and Compound 11 were administered orally to miceat a dose of 30 mg/kg. At 24 hours post dose, the mice were euthanizedwith intraperitoneal barbiturate overdose. The animals were placed in asupine position, a skin incision was made and blunt dissection followedto expose the trachea. The trachea was incised and a catheter wasinserted 4-6 mm into the trachea. Phosphate-buffered saline (PBS; 1mL/mouse) was infused into the lungs and then aspirated. Theconcentration of the BAL protein in the recovered BAL fluid wasdetermined on an ADVIA® 1800 Chemistry Analyzer (Siemens HealthcareDiagnostics). The results of the bronchoalveolar lavage (BAL) assay areshown in Table 5. The results represent the average results of allanimals within each treatment group (n=2-4).

TABLE 5 Mouse BAL protein level (mg/dL) (24 hr post-dose) RelativeExample or Dosage Compound Vehicle Control BAL protein Compound No(mg/kg) Mean SEM Mean SEM vs. control 1 30 8.25 1.0 8.3 0.33 0.99 2 308.0 0.0 8.3 0.33 0.96 6 30 14.3 2.0 7.3 1.3 1.96 8 30 7.5 1.0 7.3 1.01.03 11 30 13.3 0.5 10.0 1.0 1.33

Table 5 shows the relative BAL protein levels at 24 hours for the testedcompounds compared to the administration of vehicle only. A value forthe relative BAL protein versus control of greater than 1 indicated anincrease in pulmonary toxicity compared to the administration of vehicleonly. In this study, as reported in Table 5, the administration ofExamples 1 and 2 gave relative BAL protein levels of 0.99 and 0.96,indicating no increase in the pulmonary toxicity. The administration ofCompound 8 gave a relative BAL protein level of 1.03, which indicatedslight or no increase in pulmonary toxicity. In contrast, administrationof Compound 6 and Compound 11 gave relative BAL protein levels of 1.94and 1.33, indicating increased pulmonary toxicity.

The compounds of the present invention, as exemplified by Examples 1 and2, have been compared to a) Compounds 6 and 8, and b) ComparativeCompound 11, disclosed in WO 2008/079382, and have been found to beespecially advantageous. The compounds of the present invention had thesurprising advantage of the combination of activity in reducing bloodlymphocytes and minimizing pulmonary side effects, such as pulmonaryedema. As shown in Tables 4b and 5, in the reported tests, Examples 1and 2 of this invention show the surprising advantage in efficacy ofreducing blood lymphocytes without increasing in the BAL protein, ameasure of pulmonary side effects. For example, as compared to Compounds6, 8, and 11, the exemplified compounds of the invention reported inTable 4b and 5 reduced the blood lymphocytes by 88% and 90%, and gaverelative BAL protein levels of 0.99 and 0.96, respectively, whichindicated no increase in pulmonary side effects. In contrast, in similartests, Compound 6 and Comparative Compound 11 reduced blood lymphocytesby 78% and 52%, and gave relative BAL protein levels of 1.96 and 1.33,respectively, indicating increased risk of pulmonary side effects.Compound 8 reduced blood lymphocytes by 59% and gave relative BALprotein levels of 1.03, which indicated no or slight increase inpulmonary side effects.

TABLE 6 Mouse Blood Lymphocyte Reduction and Mouse BAL Protein Levelsfrom Tables 4b and 5 Mouse Blood Lymphocyte Mouse BAL protein levelReduction Assay at 24 hr (24 hr post-dose) Example or post-dose (Percentreduction Relative BAL protein Compound No vs. control) at 1 mg/kg vs.control at 30 mg/kg 1 88% 0.99 2 90% 0.96 6 78% 1.96 8 59% 1.03 11 52%1.33

The compounds of the present invention possess activity as agonists ofthe S1P₁ receptor, leading to the reduction of circulating bloodlymphocytes, and thus may be used in treating, preventing, or curingvarious S1P₁ receptor-related conditions while reducing or minimizingpulmonary side effects, such as pulmonary edema. The surprisingselectivity of the compounds of the present invention indicate theirpotential use in treating, preventing, or curing autoimmune andinflammatory diseases such as multiple sclerosis, rheumatoid arthritis,inflammatory bowel diseases, lupus, or psoriasis, while reducing orminimizing possible pulmonary side effects. Other potential uses of thecompounds of the present invention include minimizing or reducingrejection of transplanted organs, while reducing or minimizing possiblepulmonary side effects.

Rat Adjuvant Induced Arthritis Assay (AA)

The rat adjuvant-induced arthritis model is an animal model for humanrheumatoid arthritis.

Male Lewis rats (150-175 g; Harlan, n=8 treatment group) were immunizedat the base of the tail with 100 μl of 10 mg/ml freshly groundMycobacterium butyricum (Difco Laboratories) in incomplete Freund'sadjuvant (sigma). Animals were dosed once daily with the test article(as a solution or suspension in the vehicle) or vehicle alone(polyethylene glycol 300, “PEG300”) starting from the day ofimmunization. The volumes of their hind paws were measured in a waterdisplacement plethysmometer (Ugo Basile, Italy). The baseline pawmeasurements were taken before onset of the disease (between day 7 today 10). The paw measurements were then taken three times a week untilthe end of the study on day 20 to 21. All procedures involving animalswere reviewed and approved by the Institutional Animal Care UseCommittee.

Example 2 of the present invention was tested in the Rat AdjuvantInduced Arthritis assay described hereinabove and the results are shownin Table 7. The compound of this invention, as exemplified by Example 2,in the reported test, showed inhibition of disease progression asmeasured by reduced paw swelling in the Lewis rat using a prophylacticoral dosing regimen.

TABLE 7 Paw Swelling Group (mL) on Day 20 Vehicle Mean 1.78 SEM 0.14Example 2 Mean 1.64 (0.15 mg/kg) SEM 0.10 Example 2 Mean 0.70 (0.5mg/kg) SEM 0.13 Example 2 Mean 0.16 (1.5 mg/kg) SEM 0.04Mouse T Cell Transfer Induced Colitis Assay

The mouse T Cell transfer induced colitis assay is an animal model forhuman colitis.

Colitis was induced in CB-17 SCID mice by the adoptive transfer of FACSsorted CD4⁺CD45RB^(high) T cells from BALB/c mice (3×10⁵/mouse, i.p.).Disease activities were monitored once a week for the initial 3 weeksand 3×/week for the subsequent weeks on the basis of body weight, softstool or diarrhea, and anorectal prolapse. Animals were dosed orallyevery other day (q.o.d.) with the test article or vehicle starting fromthe day of T cell transfer. Mice were sacrificed 6 wk after T cellreconstitution and analyzed for bowel inflammation based on histologicalexamination of H&E stained colon tissues. All procedures involvinganimals were reviewed and approved by the Institutional Animal Care UseCommittee.

Example 2 was tested in the mouse T cell transfer induced colitis modeldescribed hereinabove and the results are shown in Table 8. The compoundof this invention, as exemplified by Example 2, in the reported test,showed inhibition of disease progression as measured by reduced bodyweight loss or increase body weight, and a reduction in inflammation anddamage in the mouse T Cell transfer induced colitis assay.

TABLE 8 Week 6 Percent Body Inflammation/Damage Group Weight Changes(Histology) Vehicle Mean 100% → 96.8% Mean 6.79 SD 2.4% SEM 0.36 Example2 Mean 100% → 98.7% Mean 5.46 (1 mg/kg; q.o.d.) SD 1.5% SEM 0.32 Example2 Mean  100% → 103.7% Mean 3.71 (5 mg/kg; q.o.d.) SD 1.3% SEM 0.50MRL/lpr Mouse Model of Spontaneous Lupus Erythematosus Assay

MRL/lpr mouse model of spontaneous lupus erythematosus assay is ananimal model for spontaneous lupus erythematosus.

Male MRL/lpr mice (14 weeks old; Jackson Laboratories; n=12-13) wereorally dosed with Example 2 (as a solution in vehicle) or vehicle alone(polyethylene glycol 300) twice a week for 11 weeks starting on day 0.Urine protein levels (by Albustix) were measured on day 0 and throughoutthe study. Table 9 indicates the percentage of mice in each treatmentgroups that demonstrated high levels of proteinurea (greater than 100mg/dL) at 25 weeks of age. Additional groups of mice received a dailyoral dose of dexamethasone (Dex) either independently or in combinationwith twice weekly dosing with Example 2. Urine protein levels (byAlbustix) were measured on day 0 and throughout the study. Table 9indicates the percentage of mice in each treatment groups thatdemonstrated high levels of proteinurea (greater than 100 mg/dL) at 24weeks of age. All procedures involving animals were reviewed andapproved by the Institutional Animal Care Use Committee.

Example 2 was tested in the MRL/lpr mouse model of spontaneous lupuserythematosus assay described hereinabove and the results are shown inTable 9. The compound of this invention, as exemplified by Example 2, inthe reported test, showed inhibition of disease progression as measuredby a lower percentage of mice with a proteinurea level of greater than100 mg/dL.

TABLE 9 Proteinurea at 25 Weeks of Age % Mice with Group Proteinurea >100 mg/dL Vehicle 54 Example 2 25 (0.05 mg/kg; 2x week) Example 2 17(0.4 mg/kg; 2x week) Example 2 10 (2 mg/kg; 2x week)

Example 2 and dexamethasone (Dex) either independently or incombination, were tested in the MRL/lpr mouse model of spontaneous lupuserythematosus assay described hereinabove and the results are shown inTable 10. Both the compound of this invention, as exemplified by Example2, and dexamethasone in the reported test, showed inhibition of diseaseprogression as measured by a lower percentage of mice with a proteinurealevel of greater than 100 mg/mL. In this test, no mice administered thecombination of Example 2 and dexamethasone had a proteinurea level ofgreater than 100 mg/mL.

TABLE 10 Proteinurea at 24 Weeks of Age % Mice with Group Proteinurea >100 mg/dL Vehicle 64 Dex (0.1 mg/kg q.d.) 50 Dex (0.5 mg/kg q.d.) 10Example 2 20 (2 mg/kg; 2x week) Example 2 0 (2 mg/kg; 2x week) + Dex(0.1 mg/kg q.d.)Single Crystal X-Ray Diffractometry

The single crystal data were collected on a Bruker-AXS APEX2 CCD systemusing Cu Kα radiation (λ=1.5418 Å). Indexing and processing of themeasured intensity data were carried out with the APEX2 software programsuite. When indicated, crystals were cooled in the cold stream of anOxford cryo system during data collection. The structures were solved bythe direct methods and refined on the basis of observed reflectionsusing the SHELXTL program. The derived atomic parameters (coordinatesand temperature factors) were refined through full matrix least-squares.The function minimized in the refinements was Σ_(w)(|F_(o)|−|F_(c)|)². Ris defined as Σ∥F_(o)|−|F_(c)|/Σ|F_(o)| whileR_(w)=[Σ_(w)(|F_(o)|−|F_(c)|)²/Σ_(w)|F_(o)|²]^(1/2) where w is anappropriate weighting function based on errors in the observedintensities. Typically, all the non-H atoms were refined anisotropicallyand all H-atoms other than those attached to N and O atoms werecalculated by geometrical methods and refined using a riding model.

X-Ray Powder Diffractometry

X-ray powder diffraction (PXRD) data were obtained using a Bruker GADDS(General Area Detector Diffraction System) manual chi platformgoniometer. Powder samples were placed in thin walled glass capillariesof 0.7 mm in diameter; the capillaries were rotated during datacollection. The sample-to-detector distance was kept at 17 cm. Data werecollected with Cu Kα radiation (λ=1.5418 Δ) in the range 2.5<2θ<35° witha sample exposure time of 600 seconds.

What is claimed is:
 1. A compound having the structure:

wherein said compound is in crystalline Form N-1, which is characterizedby one or more of the following: a) unit cell parameters approximatelyequal to the following: Cell dimensions: a=5.54 Å b=7.37 Å c=48.85 Åα=90.0° β=90.0° γ=90.0° Space group: P2₁2₁2₁ Molecules of saidcompound/asymmetric unit: 1 wherein the unit cell parameters of Form N-1are measured at a temperature of about −70° C.; b) a simulated powderx-ray diffraction pattern substantially as shown in FIG. 1; c) anobserved powder x-ray diffraction pattern substantially as shown FIG. 1;d) a powder x-ray diffraction pattern comprising four or more 2θ valuesselected from: 3.6±0.2, 7.2±0.2, 12.5±0.2, 14.0±0.2, 15.0±0.2, 17.5±0.2,19.4±0.2, 20.4±0.2, and 23.8±0.2, wherein the powder x-ray diffractionpattern of Form N-1 is measured at a temperature of about 25° C.; and/ore) a powder x-ray diffraction pattern comprising five or more 2θ valuesselected from: 3.6±0.2, 7.2±0.2, 12.5±0.2, 14.0±0.2, 15.0±0.2, 17.5±0.2,19.4±0.2, 20.4±0.2, and 23.8±0.2, wherein the powder x-ray diffractionpattern of Form N-1 is measured at a temperature of about 25° C.
 2. Thecompound according to claim 1, wherein said crystalline Form N-1 issubstantially pure.
 3. A pharmaceutical composition comprising thecompound in crystalline Form N-1 according to claim 1; and at least onepharmaceutically acceptable carrier and/or diluent.